RU2801948C2 - Modulation of the content of reducing sugars in the plant - Google Patents

Abstract

FIELD: biochemistry.

SUBSTANCE: invention relates to dried plant material from Nicotiana tabacum. Also disclosed is a tobacco product containing said plant material from Nicotiana tabacum. Disclosed is a method for obtaining said dried plant material from Nicotiana tabacum.

EFFECT: invention allows to effectively modulate the expression or activity of a polynucleotide or polypeptide that affects the levels of reducing sugars in dried plant material from Nicotiana tabacum.

12 cl, 1 dwg, 2 tbl, 2 ex

Description

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

The present invention discloses polynucleotide sequences of genes encoding sucrose synthase (SUS) from Nicotiana tabacum , and their variants, homologues and fragments. Also disclosed are the polypeptide sequences encoded by them and their variants, homologues and fragments. Also disclosed is the modulation of the expression of one or more NtSUS genes or the function or activity of the NtSUS polypeptide(s) encoded by them to modulate the level of reducing sugars in a plant or part thereof.

BACKGROUND OF THE INVENTION

There is a continuing need in the art to provide a tobacco material in which the release of flavoring compounds into aerosol and smoke is modified. There is also a continuing need in the art to provide a tobacco material that alters the release of acrylamide into aerosol and smoke.

The present invention is directed to meeting these and other needs.

BRIEF DESCRIPTION OF THE INVENTION

Sucrose synthase (SUS) is a key enzyme involved in the synthesis of sucrose and a key enzyme that triggers the accumulation of reducing sugars such as glucose, fructose, lactose and maltose in dried tobacco leaves. By modulating SUS expression, the pool of reducing sugars can be modulated during and after drying of the tobacco leaf. Altered reducing sugar content in dried tobacco can lead to different perceptions of tobacco flavor and aroma produced by heating. As described herein, certain NtSUS genes are overexpressed during drying, while others are not expressed and will not contribute to modulating reducing sugar levels. However, these genes are likely to be involved in other metabolic pathways and changes in their expression may result in a phenotype that is agronomically unfavorable (eg slow growth). Knowing which NtSUS genes are overexpressed during drying advantageously allows selection of plants with changes in the relevant genes only and reduces potential negative effects on other metabolic processes.

This document describes several SUS genomic polynucleotide sequences fromNicotiana tabacum, includingNtSUS1-S(SEQ ID NO: 1), NtSUS1-T(SEQ ID NO: 3), NtSUS2-S(SEQ ID NO: 5),NtSUS2-T(SEQ ID NO: 7), NtSUS3-S(SEQ ID NO: 9), NtSUS3-T(SEQ ID NO: 11), NtSUS4-S(SEQ ID NO: 13), NtSUS4-T(SEQ ID NO: 15), NtSUS5-S(SEQ ID NO: 17), NtSUS5-T(SEQ ID NO: 19), NtSUS6-S(SEQ ID NO: 21) AndNtSUS6-T(SEQ ID NO: 23). Also disclosed are corresponding predicted polypeptide sequences for NtSUS1-S (SEQ ID NO: 2), NtSUS1-T (SEQ ID NO: 4), NtSUS2-S (SEQ ID NO: 6), NtSUS2-T (SEQ ID NO: 8) , NtSUS3-S (SEQ ID NO: 10), NtSUS3-T (SEQ ID NO: 12), NtSUS4-S (SEQ ID NO: 14), NtSUS4-T (SEQ ID NO: 16), NtSUS5-S (SEQ ID NO: 18), NtSUS5-T (SEQ ID NO: 20), NtSUS6-S (SEQ ID NO: 22) and NtSUS6-T (SEQ ID NO: 24).NtSUS2-S,NtSUS2-T, NtSUS3-S,NtSUS3-T,NtSUS4-S AndNtSUS4-T may play a role in the metabolism of sugars during drying. In particular, it is shown thatNtSUS2-S,NtSUS3-S,NtSUS3-T AndNtSUS4-S play a role in the metabolism of sugars during drying. During the drying of tobacco, fire-cured tobacco typically contains at least eight times more reducing sugars than air-cured tobacco, due primarily to the high level of starch accumulation in Virginia tobacco. However, after harvesting plants and during the aging process, most starch is first converted to sucrose and then to reducing sugars, apparently involving SUS as well as invertases as a key enzyme for sucrose metabolism. Therefore, the present invention is particularly useful in modulating the content of reducing sugars in dried plant material.

In one aspect, a plant cell is described comprising: (i) a polynucleotide comprising, consisting of, or essentially consisting of a sequence having at least 95% sequence identity with SEQ ID NO: 1, or SEQ ID NO: 3, or SEQ ID NO : 9 or SEQ ID NO: 11 or SEQ ID NO: 17 or SEQ ID NO: 19 or SEQ ID NO: 21 or SEQ ID NO: 23; or a polynucleotide comprising, consisting of, or essentially consisting of a sequence having at least 80% sequence identity with SEQ ID NO: 5, or SEQ ID NO: 7, or SEQ ID NO: 13, or SEQ ID NO: 15; (ii) a polypeptide encoded by the polynucleotide shown in (i); (iii) a polypeptide comprising, consisting of, or essentially consisting of a sequence having at least 94% sequence identity with SEQ ID NO: 2; or at least 93% sequence identity with SEQ ID NO: 4; or at least 95% sequence identity with SEQ ID NO: 6; or at least 96% sequence identity with SEQ ID NO: 8; or at least 93% sequence identity with SEQ ID NO: 10 or SEQ ID NO: 12; or at least 95% sequence identity with SEQ ID NO: 14; or at least 96% sequence identity with SEQ ID NO: 16; or at least 89% sequence identity with SEQ ID NO: 18; or at least 92% sequence identity with SEQ ID NO: 20; or at least 93% sequence identity with SEQ ID NO: 22; or at least 94% sequence identity with SEQ ID NO: 24; or (iv) a construct, vector or expression vector comprising the isolated polynucleotide shown in (i), wherein said plant cell contains at least one modification that modulates the expression or activity of the polynucleotide or polypeptide compared to a control plant cell in which the expression or the activity of the polynucleotide or polypeptide has not been modified.

Also described is a polypeptide containing, consisting or essentially consisting of a sequence characterized by at least 69% sequence identity with SEQ ID NO: 2 or SEQ ID NO: 4; or at least 80% sequence identity with SEQ ID NO: 6 or SEQ ID NO: 8; or at least 74% sequence identity with SEQ ID NO: 10 or SEQ ID NO: 12; or at least 76% sequence identity with SEQ ID NO: 14 or SEQ ID NO: 16; or at least 68% sequence identity with SEQ ID NO: 18 or SEQ ID NO: 20; or at least 69% sequence identity with SEQ ID NO: 22 or SEQ ID NO: 24.

Preferably, said plant cell contains a polynucleotide comprising, consisting of, or essentially consisting of a sequence having at least 80% sequence identity with SEQ ID NO: 5 or SEQ ID NO: 13, or at least 95% sequence identity with SEQ ID NO: 9 or SEQ ID NO: 11.

Preferably, said plant cell comprises a polypeptide comprising, consisting of, or essentially consisting of a sequence having at least 81% sequence identity with SEQ ID NO: 6, or at least 72% sequence identity with SEQ ID NO: 10 or SEQ ID NO : 12, or at least 80% sequence identity with SEQ ID NO: 14.

Preferably, said plant cell comprises a polypeptide comprising, consisting of, or essentially consisting of a sequence having at least 80% sequence identity with SEQ ID NO: 6, or at least 74% sequence identity with SEQ ID NO: 10 or SEQ ID NO : 12, or at least 76% sequence identity with SEQ ID NO: 14.

Preferably, at least one modification is a modification of the genome of a plant cell, or a modification of a construct, vector or expression vector, or a transgenic modification.

Preferably, the modification of the genome of the plant cell or the modification of the construct, vector or expression vector is a mutation or editing.

Preferably, the modification increases or decreases the expression or activity of the polynucleotide or polypeptide compared to a control plant cell.

Preferably, the plant cell contains an interference polynucleotide containing a sequence that is at least 80% complementary to at least 19 nucleotides of the RNA transcribed from the polynucleotide according to claim 1(i).

Preferably, the modulated expression or activity of the polynucleotide or polypeptide modulates the level of reducing sugars in a leaf derived from a plant cell as compared to the level of reducing sugar in a leaf derived from a control plant, respectively, where the reducing sugar is glucose or fructose, or, respectively, where the sheet is a sheet subjected to the early stages of drying or a dried sheet.

In another aspect, a plant or part thereof is described that contains the plant cell described herein.

In another aspect, a plant material, a dried plant material, or a homogenized plant material obtained from the plant or part thereof described herein is described.

Preferably, the plant material comprises the biomass, seed, stem, flowers or leaves of the plant or part thereof described herein.

Preferably, the dried plant material is fire-dried, sun-dried or air-dried plant material.

In another aspect, a tobacco product is described comprising a plant cell as described herein, a plant part as described herein, or plant material as described herein.

In another aspect, a method for obtaining a plant described herein is described, comprising the following steps: (a) providing a plant cell containing a polynucleotide containing, consisting of, or essentially consisting of a sequence characterized by at least 95% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 3 or SEQ ID NO: 9 or SEQ ID NO: 11 or SEQ ID NO: 17 or SEQ ID NO: 19 or SEQ ID NO: 21 or SEQ ID NO: 23 ; or characterized by at least 80% sequence identity with SEQ ID NO: 5, or SEQ ID NO: 7, or SEQ ID NO: 13, or SEQ ID NO: 15; (b) modifying a plant cell to modulate the expression of said polynucleotide as compared to a control plant cell; and (c) propagating the plant cell to produce a plant.

Preferably, step (c) comprises cultivating a plant from a shoot or seedling containing a plant cell.

Preferably, the step of modifying the plant cell includes modifying the genome of the cell using genome editing or genomic engineering techniques.

Preferably, genome editing or genomic engineering techniques are selected from CRISPR/Cas technology, zinc finger nuclease mediated mutagenesis, chemical or radiation mutagenesis, homologous recombination, oligonucleotide directed mutagenesis, and meganuclease mediated mutagenesis.

Preferably, the step of modifying a plant cell comprises transfecting the cell with a construct comprising a polynucleotide comprising, consisting of, or essentially consisting of a sequence having at least 95% sequence identity with SEQ ID NO: 1, or SEQ ID NO: 3, or SEQ ID NO: 9 or SEQ ID NO: 11 or SEQ ID NO: 17 or SEQ ID NO: 19 or SEQ ID NO: 21 or SEQ ID NO: 23; or having at least 80% sequence identity with SEQ ID NO: 5 or SEQ ID NO: 7 or SEQ ID NO: 13 or SEQ ID NO: 15 operably linked to a constitutive promoter.

Preferably, the step of modifying the plant cell comprises introducing into the cell an interference polynucleotide containing a sequence that is at least 80% complementary to the RNA transcribed from the polynucleotide according to claim 1(i).

Preferably, a plant cell is transfected with a construct expressing an interference polynucleotide containing a sequence that is at least 80% complementary to at least 19 nucleotides of the RNA transcribed from the polynucleotide described herein.

In another aspect, a method is described for obtaining a dried plant material with a modified amount of reducing sugars compared to a control plant material, comprising the steps of: (a) providing the plant or part thereof or plant material described herein; (b) collection of plant material from them; and (c) drying the plant material.

Preferably, the plant material comprises dried leaves, dried stems or dried flowers, or a mixture thereof.

Preferably, the drying method is selected from the group consisting of air drying, flame drying, flue drying and flue drying.

SOME BENEFITS

Advantageously, the NtSUSα polynucleotide sequences described herein are expressed during drying, in particular from the start of drying. Modulating the expression of one or more NtSUS polynucleotide sequences can result in modulated levels of reducing sugars such as glucose and fructose in dried tobacco leaves. In particular, an increase or decrease in the expression of one or more NtSUS polynucleotides can lead to an increase or decrease in the levels of reducing sugars and a different perception of tobacco derived from them when heated.

In cigarette smoke, reducing sugars influence the release of carbonyl components such as formaldehyde, acetaldehyde, acetone, 2-furfural and acrolein (see The Chemical Components of Tobacco and Tobacco Smoke, ^2nd Edition, Rodgman and Perfetti, 2016). Therefore, changing the content of reducing sugars can change the release of such carbonyl compounds. With respect to flavor release in aerosol and smoke, glucose and to a lesser extent fructose can form Amadori compounds via the Maillard reaction when reacted with amino acids upon heating. Although free amino acids are considered to be the limiting factors in this chemical reaction, the presence of reducing sugars is necessary to ensure the formation of Amadori compounds. Bready, nutty, or popcorn-like flavor and aroma are produced by heating proline and glucose, thus forming products like 2-acetyl-1-pyrroline. Since proline and reducing sugars are especially common in fire-cured tobacco, the nutty notes are produced by heating or smoking Virginia tobacco. In the case of a mixture, typically acrylamide (a carcinogenic compound resulting from the reaction of glucose (fructose) with asparagine when heated) can be obtained by combining Burley tobacco (high in asparagine) with Virginia tobacco (high in reducing sugars). Therefore, modification of the balance of reducing sugar and amino acid can affect the release of both flavor compounds and acrylamide in aerosol and smoke.

Advantageously, reducing sugars are often required to properly manufacture a molded sheet of recombinant tobacco material for heated tobacco. Modulating the expression of SUS genes during leaf drying can affect the content and balance of reducing sugars, thereby affecting the production of the molded sheet. Advantageously, non-genetically modified plants can be created that may be more acceptable to consumers.

Advantageously, the present invention is not limited to the use of plants obtained by EMS mutagenesis. A plant produced by EMS mutagenesis may have less potential to transfer improved traits to the crop after selection. After the start of breeding, the necessary characteristic (characteristics) of the plant obtained by EMS mutagenesis may be lost for various reasons. For example, multiple mutations may be required, a mutation may be dominant or recessive, and identification of a point mutation in a target gene may be difficult to achieve. On the contrary, the present invention introduces the use of NtAAT polynucleotides, which can in particular be manipulated to obtain plants with the desired phenotype. The present invention can be applied to various varieties of plants or crops.

BRIEF DESCRIPTION OF GRAPHICS

Figure 1 is a bar graph showing the content of reducing sugars after harvest (mature leaves), after two days of drying (48 hours of drying) and at the end of drying for each variety.

DETAILED DESCRIPTION

The section headings used in this disclosure are for organizational purposes and are not intended to be limiting.

1. Definitions

Unless otherwise defined, all technical and scientific terms used in this document have the same meaning as usually understood by a person of ordinary skill in the art. In the event of conflict, this document, including the definitions, shall prevail. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein may be used in practicing or testing the present invention. The materials, methods, and examples disclosed herein are illustrative only and are not intended to be limiting.

The terms "include(includes)", "include(includes)", "characterized", "has", "may", "comprise(contains)" and their variants used in this document are intended to be open transitions. phrases, terms or words that do not exclude the possibility of additional actions or structures.

The singular forms include references to the plural unless the context clearly indicates otherwise.

The term "and/or" means (a) or (b) or both (a) and (b).

The present invention contemplates other embodiments "comprising", "consisting of" and "essentially consisting of" the embodiments or elements presented herein, whether explicitly stated or not.

When numerical ranges are set forth in this document, each number in between is explicitly provided for with the same degree of precision. For example, in the case of the range 6-9, in addition to 6 and 9, the numbers 7 and 8 are provided, and in the case of the range 6.0-7.0, the numbers 6.0, 6.1, 6.2, 6 are explicitly provided, 3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 and 7.0.

The following terms, as used throughout this specification and claims, have the following meanings.

The terms "coding sequence" or "polynucleotide encoding" means the nucleotides (RNA or DNA molecules) that make up a polynucleotide that encodes a polypeptide. The coding sequence may further comprise initiation and termination signals operably linked to regulatory elements, including a promoter and a polyadenylation signal, capable of directing expression in cells of the individual or mammal receiving the polynucleotide. The coding sequence may be codon-optimized.

The terms "complementary sequence" or "complementary" may refer to Watson-Crick base pairing (e.g., A-T/U and C-G) or Hoogsteen between nucleotides or nucleotide analogs. "Complementarity" refers to the property shared by two polynucleotides that when they are anti-parallel aligned with each other, the nucleotide bases at each position are complementary to each other.

The term "construct" refers to a double-stranded fragment of a recombinant polynucleotide containing one or more polynucleotides. The construction contains a "matrix thread", the bases of which are paired with a complementary "semantic or coding thread". The construct can be inserted into the vector in two possible orientations: either in the same (or sense) orientation, or in the opposite (or antisense) orientation to the orientation of the promoter located in the vector, such as an expression vector.

The term "control" in the context of a control plant or control plant cells means a plant or plant cells in which the expression, function, or activity of one or more genes or polypeptides has not been modified (e.g., increased or decreased), and therefore may provide a comparison with a plant in which the expression, function or activity of one or more of the same genes or polypeptides has been modified. Used in this document, the expression "control plant" means a plant that is practically equivalent to the test plant or modified plant in all respects, with the exception of the tested parameters. For example, if we are talking about a plant in which the polynucleotide has been introduced, the control plant is an equivalent plant in which such a polynucleotide has not been introduced. The control plant may be an equivalent plant into which the control polynucleotide has been introduced. In such cases, a control polynucleotide is a polynucleotide for which its phenotypic effect on the plant is expected to be negligible or absent. The control plant may contain an empty vector. The control plant may correspond to the wild type plant. The control plant may be a null segregant, with the T1 segregant no longer containing the transgene.

The terms "donor DNA" or "donor template" refer to a double stranded DNA fragment or molecule that contains at least a portion of a gene of interest. The donor DNA may encode a fully functional polypeptide or a partially functional polypeptide.

The term "endogenous gene or polypeptide" refers to a gene or polypeptide that originates from the genome of an organism and has not undergone a change, such as loss, gain or replacement of genetic material. An endogenous gene undergoes normal gene transfer and gene expression. The endogenous polypeptide undergoes normal expression.

The term "enhancer sequences" refers to sequences that can increase gene expression. These sequences may be located upstream, within the introns, or downstream of the transcribed region. The transcribed region consists of exons and introns located between them, from the promoter to the transcription termination site. The increase in gene expression can occur through a variety of mechanisms, including increased transcriptional efficiency, stabilization of mature mRNA, and increased translation.

The term "expression" refers to the production of a functional product. For example, expression of a polynucleotide fragment may refer to the transcription of a polynucleotide fragment (eg, transcription resulting in mRNA or functional RNA) and/or translation of the mRNA to produce a precursor or mature polypeptide. "Overexpression" refers to the production of a gene product in transgenic organisms at levels that exceed those in a null-segregant (or non-transgenic) organism from the same experiment.

The terms "functional" and "fully functional" describe a polypeptide that has a biological function or activity. The term "functional gene" refers to a gene that is transcribed to form an mRNA that is translated to form a functional or active polypeptide.

The term "genetic construct" refers to DNA or RNA molecules that contain a polynucleotide that encodes a polypeptide. The coding sequence may contain initiation and termination signals operably linked to regulatory elements, including a promoter and a polyadenylation signal, capable of driving expression.

The term "genome editing" refers to such a change in an endogenous gene that encodes an endogenous polypeptide, which achieves the expression of a polypeptide that is a truncated endogenous polypeptide or an endogenous polypeptide having an amino acid substitution. Genome editing may include replacing the portion of an endogenous gene to be targeted, or replacing the entire endogenous gene with a copy of the gene that is characterized by the presence of a truncation or amino acid substitution, using a repair mechanism such as HDR. Genome editing may also include creating an amino acid substitution in an endogenous gene by creating a double strand break in the endogenous gene, which is then repaired using NHEJ. NHEJ can be used to add or delete at least one base pair during repair, whereby an amino acid substitution can be obtained. Genome editing may also include deletion of a gene segment by simultaneous action of two nucleases on the same strand of DNA to effect truncation between two nuclease target sites, and repair of the DNA break by NHEJ.

The term "heterologous" in relation to a sequence means a sequence that is derived from a foreign species or, if derived from the same species, is substantially modified from its native form in composition and/or genomic locus due to intentional human interference.

The terms "repair by homologous recombination" or "HDR" refers to the mechanism of repair of double-stranded DNA damage in cells when a homologous DNA fragment is present in the nucleus, mainly in the G2 and S phases of the cell cycle. HDR uses donor DNA or a donor template to guide repair and can be used to create specific sequence changes in the genome, including targeted addition of entire genes. If the donor template is provided together with a site-specific nuclease, then the cellular apparatus will repair the break by homologous recombination, which is magnified by several orders of magnitude in the presence of DNA cleavage. If a homologous DNA fragment is not present, then NHEJ may occur instead.

The terms "homology" or "similarity" refers to the degree of sequence similarity between two polypeptides or two polynucleotide molecules compared by sequence alignment. The degree of homology between two individual compared polynucleotides is a function of the number of identical or matching nucleotides at the compared positions.

The terms "identical" or "identity" in the context of two or more polynucleotides or polypeptides means that the sequences are characterized by the presence of a certain percentage of residues that are the same within a certain area. The percentage can be calculated by optimally aligning two sequences, comparing the two sequences within a given region, determining the number of positions where both sequences have identical residues to get the number of matching positions, dividing the number of matching positions by the total number of positions in the defined region, and multiplying result by 100 to obtain a percentage of sequence identity. In cases where the two sequences are of different lengths, or the alignment creates one or more non-symmetrically spaced ends, and a certain comparison region includes only one sequence, then the residuals of the single sequence are included in the denominator and not in the numerator in the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. The identity can be determined by yourself or by using a computer sequence algorithm such as ClustalW, ClustalX, BLAST, FASTA, or the Smith-Waterman algorithm. The popular ClustalW multiple alignment program ( Nucleic Acids Research (1994) 22, 4673-4680; Nucleic Acids Research (1997), 24, 4876-4882) is a suitable method for generating multiple polypeptide or polynucleotide alignments. Suitable parameters for ClustalW may be as follows. For polynucleotide alignments: gap opening penalty=15.0, gap extension penalty=6.66 and template=identity. For polypeptide alignments: gap opening penalty=10.0, gap extension penalty=0.2 and matrix=Gonnet. For DNA and protein alignments: ENDGAP=-1 and GAPDIST=4. Those skilled in the art will appreciate that it may be necessary to vary these and other parameters for optimal sequence alignment. Preferably, the percentage of identities is then calculated based on this alignment in the form (N/T), where N is the number of positions at which an identical residue is present in the sequences, and T is the total number of positions compared, including gaps but excluding ledges.

The terms "increase" or "increased" refer to an increase of from about 10% to about 99%, or an increase of at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least at least 98%, at least 99%, at least 100%, at least 150%, or at least 200%, or more, of an amount or function or activity, such as, without limitation, the function or activity of a polypeptide, transcriptional function or activity and/or expression of the protein. The term "increased" or the phrase "increased amount" may refer to an amount or function or activity of a modified plant or product derived from a modified plant that is greater than would be found in a plant or product from the same plant variety treated with the same in a manner that has not been modified. Thus, in some cases, a wild-type plant of the same variety that has been processed in the same way is used as a control by which it is measured whether an increase in quantity has been achieved.

The term "increase" or "increased" as used herein refers to a decrease of from about 10% to about 99%, or a decrease of at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100%, or at least 150%, or at least 200% or more of an amount or function, such as polypeptide function, transcriptional function, or expression polypeptide. The term "increased" or the phrase "increased amount" may refer to an amount or function in a modified plant or product derived from a modified plant that is less than would be found in a plant or product from the same plant variety treated in the same manner that has not been modified. Thus, in some cases, a wild-type plant of the same variety that has been processed in the same way is used as a control to measure whether a reduction has been achieved.

The term "inhibit" or "inhibited" refers to a reduction of from about 98% to about 100%, or a reduction of at least 98%, at least 99%, and in particular 100%, of the amount, or function, or activity, such as, without limitation, function or activity of the polypeptide, transcriptional function or activity, and/or expression of the polypeptide.

The term "introduced" means the delivery of a polynucleotide (eg, construct) or polypeptide into a cell. The expression "introduced" includes reference to the insertion of a polynucleotide into a eukaryotic cell, where the polynucleotide may be inserted into the cell's genome, and also includes a reference to the transient introduction of the polynucleotide or polypeptide into the cell. The expression "introduced" includes references to methods of stable or transient transformation, as well as to sexual interbreeding. Thus, the expression "introduced" in relation to the insertion of a polynucleotide (e.g., recombinant construct/expression construct) into a cell means "transfection" or "transformation" or "transduction" and includes reference to the insertion of the polynucleotide into a eukaryotic cell, where the polynucleotide may be integrated into the cell genome (eg, chromosomal, plasmid, plastid, or mitochondrial DNA), converted to an autonomous replicon, or transiently expressed (eg, transfected mRNA).

The terms "isolated" or "purified" refer to material that is substantially or essentially free of the components that normally accompany it, as found in its native state. Typically, purity and homogeneity are determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The polypeptide, which is the predominant molecule present in the formulation, is substantially purified. In particular, the isolated polynucleotide is separated from the open reading frames that flank the desired gene and encode polypeptides other than the desired polypeptide. The term "purified" as used herein means that the polynucleotide or polypeptide yields substantially one band on an electrophoretic gel. In particular, this means that the polynucleotide or polypeptide is at least 85% pure, more preferably at least 95% pure, and most preferably at least 99% pure. Isolated polynucleotides can be purified from the host cell in which they naturally occur. Conventional nucleic acid purification methods known to those skilled in the art can be used to obtain isolated polynucleotides. The term also encompasses recombinant polynucleotides and chemically synthesized polynucleotides.

The terms "modulate" or "modulating" refer to providing or facilitating a qualitative or quantitative change, adjustment or modification of a method, pathway, function or activity of interest. Without limitation, such a change, adjustment or modification may be an increase or decrease in the level of the corresponding process, pathway, function or activity of interest. For example, gene expression or polypeptide expression or the function or activity of a polypeptide can be modulated. Typically, relative change, adjustment, or modification is determined by comparison with a control.

The term "non-homologous end-joining (NHEJ) pathway" as used herein refers to a pathway by which DNA double-strand breaks are repaired by direct ligation of the break ends without the need for a homologous template. Template-independent religation of DNA ends by NHEJ is a stochastic, error-prone repair process in which random microinsertions and microdeletions (inserts/deletions) are introduced at the DNA break point. This method can be used to deliberately destroy, delete or correct the reading frame in the sequences of target genes. Typically, NHEJ uses short homologous DNA sequences called microhomologues to guide repair. These microhomologues are often present in single strand protrusions at the ends of double strand breaks. If the overhangs are perfectly compatible, NHEJ will usually repair the gap accurately, although there may also be inaccurate repair resulting in loss of nucleotides, but it is much more common for the overhangs to be mismatched.

The term "non-naturally occurring" describes an entity such as a polynucleotide, genetic mutation, polypeptide, plant, plant cell, and plant material that is not naturally occurring or does not exist in nature. Such non-naturally occurring objects or artificial objects can be created, synthesized, initiated, modified, tampered with or manipulated in the ways described herein or as known in the art. Such non-naturally occurring objects or man-made objects can be created, synthesized, initiated, modified, tampered with or manipulated by humans. Thus, by way of example, a non-naturally occurring plant, a non-naturally occurring plant cell, or a non-naturally occurring plant material can be created using conventional plant breeding techniques such as backcrossing or gene manipulation techniques such as using antisense RNA, interfering RNA, meganuclease, and the like. As a further example, a non-naturally occurring plant, non-naturally occurring plant cell, or non-naturally occurring plant material can be created by introgression or by transferring one or more genetic mutations (e.g., one or more polymorphisms) from the first plant or plant cell. to a second plant or plant cell (which may themselves be naturally occurring), such that the resulting plant, plant cell, or plant material, or progeny thereof, contains a genetic structure (e.g., genome, chromosome, or segment thereof) that is not produced naturally, or which does not exist in nature. The resulting plant, plant cell or plant material is thus artificial or not naturally occurring. Accordingly, an artificial or non-naturally occurring plant or plant cell can be generated by modifying a genetic sequence in a first naturally occurring plant or plant cell, even if the resulting genetic sequence occurs naturally in a second plant or plant cell that contains a different genetic background than that of the first plant or plant cell. In certain embodiments, the mutation is not a naturally occurring mutation that occurs naturally in a polynucleotide or polypeptide, such as a gene or polypeptide. Differences in genetic background can be detected by phenotypic differences or by molecular biology techniques known in the art, such as polynucleotide sequencing, determining the presence or absence of genetic markers (eg microsatellite RNA markers).

The terms "oligonucleotide" or "polynucleotide" means at least two nucleotides covalently linked together. The description of an individual strand also defines the sequence of the complementary strand. Thus, the polynucleotide also spans a strand complementary to the single strand described. Many variants of the polynucleotide can be used for the same purpose as the specified polynucleotide. Thus, a polynucleotide also encompasses substantially identical polynucleotides and their complementary sequences. A single strand is a probe that can hybridize to a given sequence under stringent hybridization conditions. Thus, the polynucleotide also encompasses a probe that hybridizes under stringent hybridization conditions. Polynucleotides may be single or double stranded, or may contain portions of both double and single stranded sequences. The polynucleotide may be DNA, either genomic or cDNA, RNA, or a hybrid molecule, where the polynucleotide may contain combinations of deoxyribo- and ribonucleotides, as well as combinations of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine and isoguanine. Polynucleotides can be produced by chemical synthesis methods or by recombinant methods.

The specificity of single-stranded DNA for hybridization with complementary fragments is determined by the "stringency" of the reaction conditions (Sambrook et al ., Molecular Cloning and Laboratory Manual, Second Ed., Cold Spring Harbor (1989)). The rigidity of hybridization increases as the propensity to form DNA duplexes decreases. In polynucleotide hybridization reactions, stringency can be chosen to promote specificity (high stringency) hybridization reactions that can be used to identify, for example, full length clones from a library. Hybridization reactions with less specificity (low stringency) can be used to identify related but not exactly matching (homologous but not identical) DNA molecules or segments. DNA duplexes are stabilized by (1) a certain number of complementary base pairs; (2) a certain type of base pairs; (3) concentration of salts (ionic strength) in the reaction mixture; (4) the reaction temperature; and (5) the presence of certain organic solvents, such as formamide, which reduce the stability of the DNA duplex. Generally, the longer the probe, the higher the temperature required for proper annealing. The generally accepted approach is to change the temperature; higher relative temperatures lead to more severe reaction conditions. For "stringent conditions" hybridization, hybridization protocols have been described in which polynucleotides with at least 60% homology to one another remain hybridized. Generally, stringent conditions are chosen such that the temperature is approximately 5° C. lower than the melting point (Tm) temperature for a particular sequence at certain ionic strengths and pH. Tm is the temperature (at certain values of ionic strength, pH, and polynucleotide concentration) at which 50% of probes complementary to a given sequence hybridize to that sequence at equilibrium. Since these sequences are usually present in excess, at Tm 50% of the probes are occupied in the equilibrium state.

"Stringent hybridization conditions" are conditions that allow a probe, primer, or oligonucleotide to hybridize only to its particular sequence. Stringent conditions are sequence dependent and will vary. Stringent conditions typically include: (1) low ionic strength and high temperature washes, eg 15 mM sodium chloride, 1.5 mM sodium citrate, 0.1% sodium dodecyl sulfate at 50°C; (2) the presence of a denaturing agent during hybridization, e.g. 50% (v/v) formamide, 0.1% bovine serum albumin, 0.1% ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer (750 mM sodium chloride, 75 mM sodium citrate; pH 6 ,5) at 42°C or (3) the presence of 50% formamide. Typically, washes also include 5x SSC (0.75 M NaCl, 75 mM sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5x Denhardt's solution, salmon milt DNA, sonicated (50 µg/mL), 0.1% SDS and 10% dextran sulfate at 42°C with a wash at 42°C in 0.2 x SSC (sodium chloride/sodium citrate) and 50% formamide at 55° C followed by a high stringency wash with 0.1 x SSC containing EDTA at 55°C. Preferably, conditions are such that sequences that are at least about 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% homologous to each other typically remain hybridized to each other.

Under "moderate stringency conditions", wash solutions and hybridization conditions are used that are less stringent, such that the polynucleotide hybridizes to all of the polynucleotide in question, its fragments, derivatives, or analogs. One example involves hybridization in 6x SSC, 5x Denhardt's solution, 0.5% SDS, and 100 µg/ml denatured DNA from salmon milt at 55°C followed by one or more washes in 1x SSC, 0.1% SDS at 37 °C. Temperature, ionic strength, etc. can be adjusted to match experimental factors such as probe length. Other conditions of moderate stringency have been described (see Ausubel et al., Current Protocols in Molecular Biology, Volumes 1-3, John Wiley & Sons, Inc., Hoboken, NJ (1993); Kriegler, Gene Transfer and Expression: A Laboratory Manual , Stockton Press, New York, NY (1990); Perbal, A Practical Guide to Molecular Cloning, ^2nd edition, John Wiley & Sons, New York, NY (1988)).

"Low stringency conditions" use wash solutions and hybridization conditions that are less stringent than those for moderate stringency, such that the polynucleotide hybridizes to all of the polynucleotide in question, its fragments, derivatives, or analogs. A non-limiting example of low stringency hybridization conditions include hybridization in 35% formamide, 5x SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg/ml denatured salmon milt DNA, 10% (w/v) dextran sulfate at 40°C followed by one or more washes in 2x SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA and 0.1% SDS at 50°C. Other conditions of low stringency are well described, such as conditions for interspecific hybridization variants (see Ausubel et al., 1993; Kriegler, 1990).

The term "operably linked" means that the expression of a gene is under the control of the promoter to which it is spatially linked. A promoter may be located 5' (above) or 3' (below) from a gene that is under its control. The distance between a promoter and a gene may be about the same as the distance between that promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, changing this distance can be done in concert without loss of promoter function. The term "operably linked" refers to the association of polynucleotide fragments in one fragment in such a way that the function of one is regulated by the other. For example, a promoter is operably linked to a polynucleotide fragment if it is capable of regulating transcription of that polynucleotide fragment.

The term "plant" refers to any plant at any stage of its life cycle or development and its descendants. In one embodiment, the plant is a tobacco plant that belongs to a plant belonging to the genus Nicotiana. The term includes reference to whole plants, plant organs, plant tissues, plant shoots, plant seeds, and plant cells and their progeny. Plant cells include, without limitation, cells from seeds, suspension cultures, embryos, meristematic sites, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores. Suitable species, cultivars, hybrids and varieties of tobacco plants are described herein.

The terms "polynucleotide", "polynucleotide sequence" or "polynucleotide fragment" are used interchangeably herein and refer to an RNA or DNA polymer that is single or double stranded and optionally contains synthetic, non-natural or adjusted nucleotide bases. Nucleotides (usually in the form of their 5'-monophosphate) are designated using their one-letter designations as follows: "A" for adenylate or deoxyadenylate (respectively for RNA or DNA), "C" for cytidylate or deoxycytidylate, "G" for guanylate or deoxyguanylate, "U" for uridylate, "T" for deoxythymidylate, "R" for purines (A or G), "Y" for pyrimidines (C or T), "K" for G or T, "H" for A, or C or T, "I" for inosine and "N" for any nucleotide. The polynucleotide can be, without limitation, genomic DNA, complementary DNA (cDNA), mRNA, or antisense RNA, or fragment(s) thereof. In addition, the polynucleotide may be single or double stranded, a mixture of single and double stranded regions, a hybrid molecule containing DNA and RNA, or a hybrid molecule with a mixture of single and double stranded regions or fragment(s) thereof. Additionally, the polynucleotide may be composed of three-stranded sections containing DNA, RNA, or both, or fragment(s). The polynucleotide may contain one or more modified bases such as phosphothioates and may be a peptide nucleic acid (PNA). Typically, polynucleotides can be assembled from isolated or cloned cDNA fragments, genomic DNA, oligonucleotides, or single nucleotides, or a combination of the above. Although the polynucleotides described herein are presented as DNA sequences, the polynucleotides include their respective RNA sequences and their complementary (eg, fully complementary) DNA or RNA sequences, including their reverse complementary strands. The polynucleotides of the present invention are shown in the accompanying sequence listing.

The terms "polypeptide" or "polypeptide sequence" refer to a polymer of amino acids in which one or more amino acid residues are an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring polymers of amino acids. The terms also include modifications including, but not limited to, glycosylation, lipid addition, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, and ADP-ribosylation. The polypeptides of the present invention are presented in the attached sequence listing.

The term "promoter" means a synthetic or naturally derived molecule that is capable of providing, activating or enhancing the expression of a polynucleotide in a cell. The term refers to an element/sequence of a polynucleotide located, as a rule, higher in sequence and functionally linked to a fragment of a double-stranded polynucleotide. Promoters may be derived entirely from regions near the native gene of interest, or may be composed of different elements derived from different native promoters or synthetic polynucleotide segments. The promoter may contain one or more specific transcription control sequences to further enhance expression and/or adjust expression spatially and/or temporally. The promoter may also contain distal enhancer or repressor elements, which may be located up to several thousand base pairs from the transcription start site. The promoter can be obtained from sources including viruses, bacteria, fungi, plants, insects and animals. A promoter may regulate the expression of a gene component constitutively or differentially with respect to the cell, tissue, or organ in which expression occurs, or with respect to the developmental stage at which expression occurs, or in response to external stimuli such as physiological stress, pathogens, ions. metals or inducing agents.

The terms "tissue-specific promoter" and "tissue-preferred promoter", used interchangeably herein, refer to a promoter that is predominantly, but not necessarily exclusively, expressed in one organ or tissue, but may also be expressed in one particular cell. The phrase "developmentally regulated promoter" refers to a promoter whose function is determined by developmental events. The expression "constitutive promoter" refers to a promoter that causes the expression of a gene in most cell types in most cases. An "inducible promoter" provides for the selective expression of an operably linked DNA sequence in response to the presence of endogenous or exogenous stimuli, e.g. via chemical compounds (chemical inducers) or in response to environmental, hormonal, chemical and/or related signals. development. Examples of inducible or regulated promoters include promoters regulated by light, heat, stress, flood or drought, pathogens, phytohormones, wounds, or chemicals such as ethanol, jasmonate, salicylic acid, or antidotes.

The term "recombinant" as used herein refers to an artificial combination of two otherwise separated sequence segments, obtained, for example, by chemical synthesis or by manipulation of isolated polynucleotide segments using genetic engineering techniques. The term also includes reference to a cell or vector that has been modified by introducing a heterologous polynucleotide, or a cell derived from a cell so modified, but does not encompass modification of the cell or vector as a result of naturally occurring events (e.g., spontaneous mutation, natural transformation). , or transductions, or transpositions), such as those that occur without intentional human intervention.

The term "recombinant construct" refers to a combination of polynucleotides that do not normally occur together in nature. Accordingly, a recombinant construct may contain regulatory sequences and coding sequences obtained from different sources, or regulatory sequences and coding sequences obtained from the same source but arranged differently than would normally occur in nature. The recombinant construct may be a recombinant DNA construct.

The terms "regulatory sequences" and "regulatory elements", used interchangeably herein, refer to polynucleotide sequences located upstream (5' non-coding sequences), within or downstream (3' non-coding sequences) of a coding sequence that affect transcription. , processing or stability of the RNA or translation of the associated coding sequence. Regulatory sequences include promoters, translational leader sequences, introns, and polyadenylation recognition sequences. The terms "regulatory sequence" and "regulatory element" are used interchangeably herein.

The expression "site-specific nuclease" refers to an enzyme capable of specifically recognizing and cleaving DNA sequences. The site-specific nuclease may be engineered. Examples of engineered site-specific nucleases include zinc finger nucleases (ZFNs), TAL effector nucleases (TALENs), CRISPR/Cas9 based systems, and meganucleases.

The term "tobacco" is used collectively to refer to tobacco crops (e.g., a variety of field-grown and non-hydroponic tobacco plants), tobacco plants, and parts thereof, including but not limited to roots, stems, leaves, flowers. and seeds prepared and/or obtained as described herein. It is understood that "tobacco" includes Nicotiana tabacum plants and products thereof.

The term "tobacco products" refers to consumer tobacco products, including, without limitation, smoking materials (e.g., cigarettes, cigars, and pipe tobacco), snuff, chewing tobacco, chewing gum, and lozenges, and components, materials, and ingredients. for the production of consumer tobacco products. Preferably, these tobacco products are made from tobacco leaves and stems harvested from tobacco and cut, dried, dried and/or fermented in accordance with conventional tobacco production techniques.

The terms "transcriptional terminator", "termination sequences", or "terminator" refer to DNA sequences downstream of a coding sequence, including recognizable polyadenylation sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression. The polyadenylation signal is typically characterized by the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor.

The term "transgenic" refers to any cell, cell line, callus, tissue, plant part, or plant whose genome has been altered as a result of the presence of a heterologous polynucleotide, such as a recombinant construct, including the original transgenic events, as well as those obtained by procedures sexual interbreeding or asexual reproduction from the original transgenic object. The term does not cover the adjustment of the genome (chromosomal or extrachromosomal) by conventional plant breeding techniques or by naturally occurring events such as accidental cross-pollination, infection by a non-recombinant virus, transformation by non-recombinant bacteria, non-recombinant transposition, or spontaneous mutation.

The expression "transgenic plant" refers to a plant that contains in its genome one or more heterologous polynucleotides, i.e. a plant that contains recombinant genetic material not normally found in it, and which has been introduced into the plant in question (or into the plant's ancestors) through human manipulation. For example, a heterologous polynucleotide may be stably integrated into the genome such that the polynucleotide is passed on to subsequent generations. The heterologous polynucleotide may be integrated into the genome alone or as part of a recombinant construct. The commercial development of genetically improved germplasm has also progressed to the stage of introducing several traits into cultivated plants, often referred to as the gene pyramiding approach. In this approach, several genes can be introduced into a plant, conferring different characteristics of interest. Gene pyramiding can be accomplished in many ways, including, but not limited to, co-transformation, re-transformation, and crossing lines with different transgenes. Thus, a plant grown from a cell of a plant into which recombinant DNA is introduced by transformation is a transgenic plant, as is all progeny of that plant that contains the introduced transgene (either sexually or asexually). It is understood that the term "transgenic plant" encompasses the entire plant or tree and parts of the plant or tree, such as grains, seeds, flowers, leaves, roots, fruits, pollen, stems, and the like. Each heterologous polynucleotide can confer a distinct trait on the transgenic plant.

The term "transcriptional activator-like effector" or "TALE" refers to a polypeptide structure that recognizes and binds to a specific DNA sequence. The expression "TALE DNA binding domain" refers to a DNA binding domain that contains an array of 33-35 amino acid tandem repeats, also known as RVD modules, each of which specifically recognizes one DNA base pair. RVD modules can be arranged in any order, gathering in an array that recognizes a certain sequence. The binding specificity of the TALE DNA-binding domain is determined by the RVD array followed by a single truncated repeat of 20 amino acids. The DNA binding domain of TALE can have 12 to 27 RVD modules, each containing an RVD and recognizing one DNA base pair. Specific RVDs have been identified that recognize each of the four possible DNA nucleotides (A, T, C and G). Because the DNA-binding domains of TALE are modular, the repeats that recognize four different DNA nucleotides can be joined together to recognize any particular DNA sequence. These targeted DNA binding domains can then be combined with catalytic domains to create functional enzymes, including artificial transcription factors, methyltransferases, integrases, nucleases, and recombinases.

The terms "effector nucleases like transcription activators" or "TALEN", used interchangeably herein, refer to engineered fusion polypeptides based on the catalytic domain of a nuclease, such as Fok I endonuclease, and the designed TALE DNA-binding domain, which can be targeted to specially synthesized DNA sequence.

The expression "TALEN monomer" refers to an engineered fusion polypeptide with a nuclease catalytic domain and a designed TALE DNA-binding domain. The two TALEN monomers can be designed to target and cleave the TALEN target site.

The term "transgene" refers to a gene or genetic material containing a gene sequence that has been isolated from one organism and introduced into another organism. This non-native DNA segment may retain the ability to produce an RNA or polypeptide in the transgenic organism, or it may provide an adjustment to the normal function of the genetic code of the transgenic organism. The introduction of a transgene has the potential to change the phenotype of the organism.

The term "variant" in relation to a polynucleotide means: (i) a portion or fragment of a polynucleotide; (ii) a sequence complementary to the polynucleotide or a portion thereof; (iii) a polynucleotide substantially identical to the referenced polynucleotide or its complementary sequence; or (iv) a polynucleotide that hybridizes under stringent conditions to said polynucleotide, its complementary sequence, or its substantially identical polynucleotide.

The term "variant" in relation to a peptide or polypeptide means a peptide or polypeptide that differs in sequence by insertion, deletion or conservative amino acid substitution, but retains at least one biological function or activity. A variant may also mean a polypeptide that retains at least one biological function or activity. A conservative amino acid substitution, i.e. substitution of an amino acid for another amino acid with similar properties (for example, the degree of hydrophilicity and the distribution of charged sites) is understood in the art as usually including a slight change.

The term "variety" refers to a population of plants that have permanent characteristics that separate them from other plants of the same species. Although a variety has one or more distinguishing characteristics, it is additionally characterized by very little overall variation between individuals within that variety. The variety is often sold commercially.

The term "vector" refers to a polynucleotide delivery vehicle that contains a combination of polynucleotide components to allow transport of polynucleotides, polynucleotide constructs and polynucleotide conjugates, and the like. The vector may be a viral vector, a bacteriophage, a bacterial artificial chromosome, or a yeast artificial chromosome. The vector may be a DNA or RNA vector. Suitable vectors include episomes capable of extrachromosomal replication, such as double-stranded circular plasmids; linearized plasmids from a double-stranded nucleotide sequence; and other vectors of any origin. An "expression vector" as used herein is a polynucleotide delivery vehicle that contains a combination of polynucleotide components to allow expression of polynucleotide(s), polynucleotide constructs and polynucleotide conjugates, and the like. Suitable expression vectors include episomes capable of extrachromosomal replication, such as double-stranded circular plasmids; linearized plasmids from a double-stranded nucleotide sequence; and other functionally equivalent expression vectors of any origin. The expression vector contains at least a promoter upstream and operably linked to a polynucleotide, polynucleotide constructs, or polynucleotide conjugate, as defined below.

The term "zinc finger" refers to a polypeptide structure that recognizes and binds to DNA sequences. The zinc finger domain is the most common DNA-binding motif in the human proteome. One zinc finger domain contains approximately 30 amino acids and typically functions by binding to 3 consecutive base pairs of DNA through single amino acid side chain interactions with each base pair.

The term "zinc finger nuclease" or "ZFN" refers to a chimeric polypeptide molecule containing at least one zinc finger DNA-binding domain efficiently associated with at least one nuclease or a portion of a nuclease capable of being fully assembled to cleave DNA.

Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure will have the meanings that are commonly understood by those of ordinary skill in the art. For example, any nomenclature systems and techniques used in connection with cell or tissue culture, molecular biology, immunology, microbiology, polypeptide and polynucleotide genetics and chemistry, and hybridization as described herein are well known and widely used in the art. . The meaning and scope of the terms should be clear, but in the event of any implicit ambiguity, the definitions given in this document take precedence over any dictionary or non-document definition. In addition, unless otherwise required by context, singular terms will include the plural, and plural terms will include the singular.

2. Polynucleotides

In one embodiment, an isolated polynucleotide is provided comprising, consisting of, or essentially consisting of a sequence having at least 60% sequence identity with any of the sequences described herein, including any of the polynucleotides shown in the sequence listing. Preferably, the isolated polynucleotide contains, consists of, or essentially consists of a sequence characterized by at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70% , 75%, 80%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99% or 100% sequence identity with them.

Preferably, the polynucleotide(s) described herein encode(s) an active polypeptide that is characterized by at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97% , 98%, 99%, 100% or more of the function or activity of the polypeptide(s) shown in the sequence listing.

In another embodiment, an isolated polynucleotide is provided comprising, consisting of, or essentially consisting of, a polynucleotide having at least 60% sequence identity with SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 , SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21 or SEQ ID NO: 23, or c SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13.

Preferably, the isolated polynucleotide contains, consists of, or essentially consists of a sequence characterized by at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70% , 75%, 80%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99 .1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity with SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO : 17, SEQ ID NO: 19, SEQ ID NO: 21 or SEQ ID NO: 23, or with SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13.

Preferably, the isolated polynucleotide contains, consists of, or essentially consists of a sequence characterized by at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99, 5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity with SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21 or SEQ ID NO: 23, or with SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13.

Preferably, the isolated polynucleotide contains, consists of, or essentially consists of a sequence characterized by at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99, 5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity with SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 13, or SEQ ID NO: 15.

Preferably, the isolated polynucleotide contains, consists of, or essentially consists of a sequence characterized by at least about 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99. 4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity with SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 9 , SEQ ID NO: 11, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21 or SEQ ID NO: 23.

In another embodiment, polynucleotides are provided comprising, consisting of, or essentially consisting of polynucleotides with a significant degree of homology (i.e., sequence similarity) or a significant degree of identity with SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 , SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21 or SEQ ID NO: 23, or with SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13.

In another embodiment, fragments of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21 or SEQ ID NO: 23 with a significant degree of homology (i.e. sequence similarity) or with a significant degree of identity with them, which are characterized by at least , approximately 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 75%, 80%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99, 4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity with the corresponding fragments from SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21 or SEQ ID NO: 23.

In another embodiment, fragments from SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13 are presented with a significant degree of homology (i.e. sequence similarity) or a significant degree of identity with them, which are characterized by at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 75%, 80%, 85%, 87%, 88 %, 89%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99 .4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity with the corresponding fragments from SEQ ID NO:5, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13.

In another embodiment, polynucleotides are provided that provide a sufficient or substantial degree of identity or similarity to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21 or SEQ ID NO: 23, or with SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13, which encode a polypeptide that performs the SUS function.

In another embodiment, a polymer is provided that is a polynucleotide that contains, consists of, or essentially consists of, a polynucleotide referred to herein as SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 , SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21 or SEQ ID NO: 23, or as SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13.

Preferably, the polynucleotides described herein encode members of the SUS family.

The polynucleotide described herein may include a polymer of nucleotides, which may be unmodified or modified deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Accordingly, a polynucleotide may be, without limitation, genomic DNA, complementary DNA (cDNA), mRNA, or antisense RNA, or fragment(s) thereof. In addition, the polynucleotide may be single or double stranded DNA, DNA that is a mixture of single and double stranded regions, a hybrid molecule containing DNA and RNA, or a hybrid molecule with a mixture of single and double stranded regions or fragment(s) thereof. Additionally, the polynucleotide may be composed of three-strand sections containing DNA, RNA, or both, or a fragment (s). The polynucleotide may contain one or more modified bases such as phosphorothioates and may be a peptidic nucleic acid. Typically, polynucleotides can be assembled from isolated or cloned cDNA fragments, genomic DNA, oligonucleotides, or single nucleotides, or a combination of the above. Although the polynucleotides described herein are presented as DNA sequences, they include their respective RNA sequences and their complementary (eg, fully complementary) DNA or RNA sequences, including their reverse complementary sequences.

A polynucleotide described herein will typically contain phosphodiester linkages, although in some cases polynucleotide analogs are included that may have alternative backbones containing, for example, phosphoramidate, phosphorothioate, phosphorodithioate, or O-methylphosphoroamidite linkages; and peptide polynucleotide backbones and bonds. Other polynucleotide analogs include polynucleotides with positively charged backbones, nonionic backbones, and non-ribose backbones. Modifications of the ribose phosphate backbone can be carried out for a variety of reasons, for example, to increase the stability and half-life of such molecules in physiological media or as probes on a biochip. You can get mixtures of naturally occurring polynucleotides and analogues; alternatively, mixtures of different polynucleotide analogues and mixtures of naturally occurring polynucleotides and analogues can be prepared.

Many analogs of polynucleotides are known, including, for example, those with phosphoramidate, phosphorothioate, phosphorodithioate, or O-methylphosphoroamidite linkages and polynucleotide peptide backbones and linkages. Other polynucleotide analogs include those with positively charged backbones, nonionic backbones, and non-ribose backbones. Polynucleotides containing one or more carbocyclic sugars are also included.

Other analogs include peptide polynucleotides, which are those based on peptide analogs of polynucleotides. These backbones are substantially non-ionic under neutral conditions, in contrast to the highly charged phosphodiester backbone of naturally occurring polynucleotides. This may provide benefits. First, the peptide backbone of a polynucleotide may have improved hybridization kinetics. Peptide polynucleotides are characterized by greater changes in melting point in the presence of mismatched base pairs compared to perfectly matched base pairs. DNA and RNA, as a rule, are characterized by a decrease in the melting point by 2-4 C in the presence of an internal mismatch. In the case of a non-ionic peptide backbone of a polynucleotide, the drop is closer to 7-9°C. Similarly, due to their non-ionic nature, the hybridization of bases attached to these backbones is relatively insensitive to salt concentration. Additionally, peptide polynucleotides may not be degraded or degraded to a lesser extent by cellular enzymes, and thus may be more stable.

Among the uses of the disclosed polynucleotides and fragments thereof is the use of the fragments as probes in hybridization based assays or as primers for use in amplification based assays. Such fragments typically contain at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more contiguous nucleotides from the DNA sequence. In other embodiments, the DNA fragment contains at least about 10, 15, 20, 30, 40, 50, or 60 or more contiguous nucleotides from the DNA sequence. Thus, according to one aspect, a method for detecting a polynucleotide is also provided, comprising the use of probes or primers, or both. Illustrative primers are described in this document.

The main parameters influencing the choice of hybridization conditions and guidance for developing suitable conditions are described in Sambrook, J., E. F. Fritsch, and T. Maniatis (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. ). Using the genetic code data in combination with the polypeptide sequences described herein, sets of degenerate oligonucleotides can be obtained. Such oligonucleotides are useful as primers, for example, in polymerase chain reactions (PCR) by which DNA fragments are isolated and amplified. In certain embodiments, degenerate primers can be used as probes for genetic libraries. Such libraries include cDNA libraries, genomic libraries, and even electronic label libraries for expressed sequences or DNA. Homologous sequences identified by this method will then be used as probes to identify homologues of the sequences specified herein.

Also potentially useful are polynucleotides and oligonucleotides (e.g., primers or probes) that hybridize under conditions of reduced stringency, typically medium stringency, and typically high stringency, to the polynucleotide(s) described herein. document. The main parameters influencing the choice of hybridization conditions and guidance for developing suitable conditions are set forth in Sambrook J., E. F. Fritsch and T. Maniatis (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) and can be easily determined by those of ordinary skill in the art based on, for example, the length or base composition of the polynucleotide.

This document defines one way to achieve conditions of moderate and high stringency. It should be understood that the wash temperature and salt concentration in the wash as needed can be adjusted to achieve the desired degree of stringency by applying the fundamental principles that govern hybridization reactions and duplex stability, as is known to those skilled in the art and further described below (see e.g. , Sambrook, J., E. F. Fritsch, and T. Maniatis (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) When a polynucleotide is hybridized to a polynucleotide of unknown sequence, the length of the hybrid is assumed to be such as a hybridizing polynucleotide.When polynucleotides are hybridized to known sequences, the length of the fusion can be determined by aligning the sequences of the polynucleotides and identifying the site or sites with optimal sequence complementarity.The hybridization temperature for hybrids expected to be less than 50 base pairs in length should be 5 -10 C less than the melting point of the hybrid, where the melting point is determined in accordance with the following equations. For hybrids less than 18 bp in length, melting point (°C) = 2(number of bases A+T)+4(number of bases G+C). For hybrids longer than 18 bp, melting point (°C) = 81.5+16.6(log10 [Na+])+0.41(% G+C)-(600/N) where N is the number of bases in the hybrid, and [Na+] is the concentration of sodium ions in the hybridization buffer ([Na+] for 1x standard sodium citrate=0.165 M). Typically, each such hybridizing polynucleotide has a length that is at least 25% (typically at least 50%, 60%, or 70%, and most often at least 80%) of the length of the polynucleotide to which it hybridizes and is characterized at least 60% sequence identity (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) with a polynucleotide, with with which it hybridizes.

As will be appreciated by one of skill in the art, linear DNA has two possible orientations: the 5'-3' direction and the 3'-5' direction. For example, if the first sequence is located in the 5'-3' direction, and if the second sequence is located in the 5'-3' direction in the same polynucleotide molecule/strand, then the first sequence and the second sequence are oriented in the same direction or have the same orientation. . Typically, the promoter sequence and the gene of interest under the control of that promoter are in the same orientation. However, if with respect to the first sequence located in the 5'-3' direction, the second sequence is located in the 3'-5' direction in the same polynucleotide molecule/strand, then the first sequence and the second sequence are oriented in the antisense direction or have an antisense orientation. Two sequences having antisense orientations with respect to each other can alternatively be described as having the same orientation if the first sequence (the 5'-3' direction) and a sequence inversely complementary to the first sequence (the first sequence located 5'-3' ) are located within a single polynucleotide molecule/strand. The sequences set forth herein are shown in the 5'-3' direction.

At least one modification (eg, mutation) may be included in one or more of NtSUS1-S, NtSUS1-T, NtSUS2-S, NtSUS2-T, NtSUS3-S, NtSUS3-T, NtSUS4-S, NtSUS4-T, NtSUS5-S, NtSUS5-T, NtSUS6-S and NtSUS6-T. At least one modification (eg, mutation) may be included in one or more of NtSUS2-S, NtSUS2-T, NtSUS3-S, NtSUS3-T, NtSUS4-S, and NtSUS4-T .

At least one modification (e.g., a mutation) may be included in one or more of NtSUS2-S, NtSUS2-T, NtSUS3-S, NtSUS3-T, NtSUS4-S, and NtSUS4-T , with the modification(s) (e.g. , the mutation(s)) is not included in one or more of NtSUS1-S, NtSUS1-T, NtSUS5-S, NtSUS5-T, NtSUS6-S and NtSUS6-T .

At least one modification (e.g., a mutation) may be included in one or more of NtSUS2-S, NtSUS2-T, NtSUS3-S, NtSUS3-T, NtSUS4-S, and NtSUS4-T , wherein the modification(s) (e.g. , the mutation(s)) is not included in one or more of NtSUS1-S, NtSUS1-T, NtSUS5-S, NtSUS5-T, NtSUS6-S and NtSUS6-T .

At least one modification (e.g., mutation) may be included in one or more of NtSUS2-S, NtSUS3-S, NtSUS3-T, and NtSUS4-S with no modification(s) (e.g., mutation(s)) included. (included) in one or more of NtSUS1-S, NtSUS1-T, NtSUS2-T, NtSUS4-T, NtSUS5-S, NtSUS5-T, NtSUS6-S, and NtSUS6-T and they are not modulated.

At least one modification (e.g., mutation) may be included in one or more of NtSUS2-S, NtSUS3-S, NtSUS3-T, and NtSUS4-S with no modification(s) (e.g., mutation(s)) included. (included) in one or more of NtSUS1-S, NtSUS1-T, NtSUS2-T, NtSUS4-T, NtSUS5-S, NtSUS5-T, NtSUS6-S, and NtSUS6-T, and they are not modulated.

3. Polypeptide

In another aspect, an isolated polypeptide is provided, comprising, consisting of, or essentially consisting of, a polypeptide having at least 60% sequence identity with any of the polypeptides described herein, including any of the polypeptides listed in the sequences. Preferably, the isolated polypeptide contains, consists of, or essentially consists of a sequence characterized by at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 75%, 80%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, 99.1 %, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity with them.

In one embodiment, a polypeptide is provided comprising, consisting of, or essentially consisting of a sequence characterized by at least 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78% , 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99 .8%, 99.9% or 100% sequence identity with SEQ ID NO: 2 or SEQ ID NO: 4.

In another embodiment, a polypeptide is provided comprising, consisting of, or essentially consisting of a sequence characterized by at least 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99 .3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity with SEQ ID NO: 2.

In another embodiment, a polypeptide is provided comprising, consisting of, or essentially consisting of a sequence characterized by at least 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2 %, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity with SEQ ID NO: 4.

In one embodiment, a polypeptide is provided comprising, consisting of, or essentially consisting of a sequence characterized by at least 80%, 81%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99 .4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity with SEQ ID NO: 6 or SEQ ID NO: 8.

In another embodiment, a polypeptide is provided comprising, consisting of, or essentially consisting of a sequence characterized by at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3% , 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity with SEQ ID NO: 6.

In another embodiment, a polypeptide is provided comprising, consisting of, or essentially consisting of a sequence characterized by at least 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99, 4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity with SEQ ID NO: 8.

In one embodiment, a polypeptide is provided comprising, consisting of, or essentially consisting of a sequence characterized by at least 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83% , 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99 .1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity with SEQ ID NO: 10 or SEQ ID NO: 12.

In another embodiment, a polypeptide is provided comprising, consisting of, or essentially consisting of a sequence characterized by at least 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2 %, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity with SEQ ID NO: 10 or SEQ ID NO: 12.

In one embodiment, a polypeptide is provided comprising, consisting of, or essentially consisting of a sequence characterized by at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85% , 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99, 2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity with SEQ ID NO: 14 or SEQ ID NO: 16.

In another embodiment, a polypeptide is provided comprising, consisting of, or essentially consisting of a sequence characterized by at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3% , 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity with SEQ ID NO: 14.

In another embodiment, a polypeptide is provided comprising, consisting of, or essentially consisting of a sequence characterized by at least 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99, 4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity with SEQ ID NO: 16.

In one embodiment, a polypeptide is provided comprising, consisting of, or essentially consisting of a sequence characterized by at least 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77% , 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7 %, 99.8%, 99.9% or 100% sequence identity with SEQ ID NO: 18 or SEQ ID NO: 20.

In another embodiment, a polypeptide is provided comprising, consisting of, or essentially consisting of a sequence characterized by at least 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100 % sequence identity to SEQ ID NO: 18.

In another embodiment, a polypeptide is provided comprising, consisting of, or essentially consisting of a sequence characterized by at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity with SEQ ID NO: 20 .

In one embodiment, a polypeptide is provided comprising, consisting of, or essentially consisting of a sequence characterized by at least 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78% , 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99 .8%, 99.9% or 100% sequence identity with SEQ ID NO: 22 or SEQ ID NO: 24.

In another embodiment, a polypeptide is provided comprising, consisting of, or essentially consisting of a sequence characterized by at least 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2 %, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity with SEQ ID NO: 22.

In another embodiment, a polypeptide is provided comprising, consisting of, or essentially consisting of a sequence characterized by at least 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99 .3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity with SEQ ID NO: 24.

In one embodiment, a polypeptide encoded by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21 or SEQ ID NO: 23; or SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13.

The polypeptide may contain sequences having a sufficient or significant degree of identity or similarity with SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, or SEQ ID NO: 24; or SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14 to perform the SUS function.

Typically, fragments of the polypeptide(s) retain some or all of the functions or activities of the full-length sequence.

As discussed herein, polypeptides also include mutant variants obtained by introducing any type of adjustment (e.g., insertion, deletion, or substitution of amino acids; changes in glycosylation state; changes that affect refolding or isomerization, three-dimensional structures, or self-association states) that may be deliberately constructed or naturally derived, provided that they still retain some or all of their functionality or activity. Preferably, the function or activity is modulated.

A deletion refers to the removal of one or more amino acids from a polypeptide. An insert refers to one or more amino acid residues introduced at a predetermined location in a polypeptide. Insertions may include insertions into the sequence of one or more amino acids. Substitution refers to the substitution of amino acids in a polypeptide with other amino acids having similar properties (such as similar hydrophobicity, hydrophilicity, antigenicity, propensity to form or break α-helical structures or β-sheet structures). Amino acid substitutions typically include a single residue, but may be grouped depending on the functional limitations imposed on the polypeptide, and may range from about 1 to about 10 amino acids. Amino acid substitutions are preferably conservative amino acid substitutions, as described below. Amino acid substitutions, deletions and/or insertions can be performed using peptide synthesis techniques, such as solid phase peptide synthesis, or by manipulating recombinant DNA. Methods for manipulating DNA sequences to produce substitution, insertion, or deletion variants of a polypeptide are well known in the art. A variant may be characterized by the presence of adjustments that cause a "silent" change to occur and result in a functionally equivalent polypeptide. Intentional amino acid substitutions can be made based on the similarity of residues in terms of polarity, charge, solubility, hydrophobicity, hydrophilicity and amphipathic nature, provided that the binding that causes the secondary structure of the substance is maintained. For example, negatively charged amino acids include aspartic acid and glutamic acid, positively charged amino acids include lysine and arginine, and amino acids with uncharged polar end groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine and tyrosine. Conservative substitutions can be made, for example, in accordance with the table below. Amino acids in the same block in the second column, and preferably in the same row in the third column, can be substituted for one another:

ALIPHATIC non-polar Gly Ala Pro Ile Leu Val Polar - uncharged Cys Ser Thr Met Asn Gly Polar - charged AspGlu Lys Arg AROMATIC His Phe Trp Tyr

The polypeptide may be a mature polypeptide, or an immature polypeptide, or a polypeptide derived from an immature polypeptide. The polypeptides may be in linear form or be cyclized using known methods. Polypeptides typically contain at least 10, at least 20, at least 30, or at least 40 contiguous amino acids.

At least one modification (eg, mutation) may be included in one or more of NtSUS1-S, NtSUS1-T, NtSUS2-S, NtSUS2-T, NtSUS3-S, NtSUS3-T, NtSUS4-S, NtSUS4-T, NtSUS5-S, NtSUS5-T, NtSUS6-S and NtSUS6-T.

At least one modification (e.g., mutation) may be included in one or more of NtSUS2-S, NtSUS2-T, NtSUS3-S, NtSUS3-T, NtSUS4-S, and NtSUS4-T, wherein the modification(s) (e.g. , the mutation(s)) is not included in one or more of NtSUS1-S, NtSUS1-T, NtSUS5-S, NtSUS5-T, NtSUS6-S, and NtSUS6-T.

At least one modification (e.g., mutation) may be included in one or more of NtSUS2-S, NtSUS2-T, NtSUS3-S, NtSUS3-T, NtSUS4-S, and NtSUS4-T, wherein the modification(s) (e.g. , the mutation(s)) is not included in one or more of NtSUS1-S, NtSUS1-T, NtSUS5-S, NtSUS5-T, NtSUS6-S, and NtSUS6-T.

At least one modification (eg, mutation) may be included in one or more of NtSUS2-S, NtSUS3-S, NtSUS3-T, and NtSUS4-S, with no modification(s) (eg, mutation(s)) included. (included) in one or more of NtSUS1-S, NtSUS1-T, NtSUS2-T, NtSUS4-T, NtSUS5-S, NtSUS5-T, NtSUS6-S, and NtSUS6-T, and they are not modulated.

At least one modification (eg, mutation) may be included in one or more of NtSUS2-S, NtSUS3-S, NtSUS3-T, and NtSUS4-S, with no modification(s) (eg, mutation(s)) included. (included) in one or more of NtSUS1-S, NtSUS1-T, NtSUS2-T, NtSUS4-T, NtSUS5-S, NtSUS5-T, NtSUS6-S, and NtSUS6-T, and they are not modulated.

Plant modification

Transformation

Recombinant constructs can be used to transform plants or plant cells in order to modulate the expression, function, or activity of a polypeptide. The recombinant polynucleotide construct may comprise a polynucleotide encoding one or more of the polynucleotides described herein operably linked to a regulatory region suitable for expression of the polypeptide. Thus, a polynucleotide may contain a coding sequence that encodes for a polypeptide as described herein. Plants or plant cells in which the expression, function, or activity of a polypeptide is modulated may include mutant, non-naturally occurring, transgenic, human-created, or genetically engineered plants or plant cells. Preferably, the transgenic plant or plant cell contains a genome that has been corrected by stable integration of recombinant DNA. Recombinant DNA contains DNA that is genetically engineered and engineered outside of a cell, and contains DNA containing naturally occurring DNA, or cDNA, or synthetic DNA. A transgenic plant may include a plant regenerated from an originally transformed plant cell and transgenic plants that are the offspring of later generations or hybrids of the transformed plant. Preferably, the transgenic modification provides for the adjustment of the expression, or function, or activity of a polynucleotide or polypeptide described herein, compared to those in a control plant.

The polypeptide encoded by the recombinant polynucleotide may be a native polypeptide or may be heterologous to the cell. In some cases, the recombinant construct contains a polynucleotide that modulates expression operably linked to a regulatory region. Examples of suitable regulatory regions are described herein.

Also provided are vectors containing recombinant polynucleotide constructs such as those described herein. Suitable vector backbones include, for example, those commonly used in the art, such as plasmids, viruses, artificial chromosomes, bacterial artificial chromosomes, yeast artificial chromosomes, or bacteriophage artificial chromosomes. Suitable expression vectors include, but are not limited to, plasmids and viral vectors derived from, for example, bacteriophage, baculoviruses, and retroviruses. Numerous vectors and expression systems are commercially available.

The vectors may contain, for example, origins of replication, nuclear matrix binding sites, or markers. The marker gene may confer a selectable phenotype to a plant cell. For example, the marker can confer resistance to biocidal agents, such as resistance to an antibiotic (eg, kanamycin, G418, bleomycin, or hygromycin) or herbicide (eg, glyphosate, chlorsulfuron, or phosphinothricin). Additionally, the expression vector may contain a tag sequence designed to facilitate manipulation or detection (eg, purification or localization) of the expressed polypeptide. Label sequences such as luciferase, beta-glucuronidase, green fluorescent polypeptide, glutathione-S-transferase, polyhistidine, c-myc, or hemagglutinin sequences are typically expressed as a fusion fragment with the encoded polypeptide. Such labels can be inserted anywhere within the polypeptide, including at the carboxyl or amino terminus.

A plant or plant cell can be transformed by integrating the recombinant polynucleotide into its genome to ensure its stable transformation. The plant or plant cell described herein may be stably transformed. Stably transformed cells typically retain the introduced polynucleotide with each cell division. The plant or plant cell may also be transiently transformed such that the recombinant polynucleotide does not integrate into its genome. Transiently transformed cells typically lose all or some of the introduced recombinant polynucleotide with each cell division such that the introduced recombinant polynucleotide cannot be detected in daughter cells after a sufficient number of cell divisions.

A number of methods are available in the art for transforming a plant cell, including biolistics; gene gun techniques; transformation mediated by Agrobacterium; transformation mediated by a viral vector; freeze-thaw method; microparticle bombardment; direct DNA uptake; ultrasonic treatment; microinjection; plant virus-mediated transfer; and electroporation. An Agrobacterium-based system for integrating foreign DNA into plant chromosomes has been extensively studied, modified and used for plant genetic engineering. Deproteinized recombinant DNA molecules containing DNA sequences corresponding to the purified polypeptide of interest, operably linked in sense or antisense orientation to regulatory sequences, are coupled to the corresponding T-DNA sequences using conventional methods. They are introduced into protoplasts using a polyethylene glycol technique or an electroporation technique, both of which are standard. Alternatively, such vectors containing recombinant DNA molecules encoding the purified polypeptide of interest are introduced into living Agrobacterium cells, which then transfer the DNA into plant cells. Transformation with deproteinized DNA without accompanying T-DNA vector sequences can be performed by protoplast fusion with DNA-containing liposomes or by electroporation. Deproteinized DNA without accompanying T-DNA vector sequences can also be used to transform cells with inert, high speed microparticles.

If a cell or cultured tissue is used as a recipient tissue for transformation, if necessary, plants can be regenerated from the transformed cultures using methods known to those skilled in the art.

The choice of regulatory regions to be included in a recombinant construct depends on several factors, including, without limitation, potency, selectivity, inducibility, desired level of expression, and preferential expression in certain cells or tissues. It is a common task for a person skilled in the art to modulate the expression of a coding sequence through the correct selection of regulatory regions and their placement in relation to the coding sequence. Transcription of a polynucleotide can be modulated in a similar manner. Some suitable regulatory sites initiate transcription exclusively or predominantly in certain cell types. Methods for identifying and characterizing regulatory regions in plant genomic DNA are well known in the art.

Suitable promoters include tissue-specific promoters recognized by tissue-specific factors present in different tissues or cell types (eg, root-specific promoters, shoot-specific promoters, xylem-specific promoters), or present at different stages of development, or present in response to different conditions. environment. Suitable promoters include constitutive promoters that can be activated in most cell types without the need for specific inducers. Examples of suitable promoters for directing RNAi polynucleotide production include the cauliflower mosaic virus (CaMV/35S) 35S promoter, SSU, OCS, lib4, usp, STLS1, B33, nos, or ubiquitin or phaseolin gene promoters. Those skilled in the art can create many variants of recombinant promoters.

Tissue-specific promoters are transcriptional controls that are active only in certain cells or tissues at certain points in time during plant development, such as in vegetative tissues or reproductive tissues. Tissue-specific expression may be preferential, for example, if expression of polynucleotides in certain tissues is preferred. Examples of tissue-specific promoters under developmental control include promoters that can initiate transcription only (or mostly only) in certain tissues, such as vegetative tissues, e.g. roots or leaves, or reproductive tissues such as fruits, ovules, seeds, pollen, stamens, flowers or any embryonic tissue. Promoters specific for reproductive tissues can be, for example, anther specific, ovule specific, embryo specific, endosperm specific, integument specific, seed and seed coat specific, pollen specific, petal specific, sepal specific. or for combinations thereof.

Suitable leaf-specific promoters include the pyruvatortophosphate dikinase (PPDK) gene promoter from a C4 plant (maize), the cab-m1Ca+2 promoter from maize, the myb-related genes promoter from Arabidopsis thaliana (Atmyb5), ribulose biphosphate carboxylase (RBCS) promoters (e.g., tomato genes). RBCS1, RBCS2 and RBCS3A expressed in leaves and light-grown seedlings, RBCS1 and RBCS2 expressed in developing tomato fruits, or the ribulose bisphosphate carboxylase gene promoter expressed at high levels almost exclusively in mesophilic cells of leaf blades and leaf axils).

Suitable aging tissue-specific promoters include the promoter from tomato, active during fruit ripening, senescence and leaf abscission, the promoter of the gene encoding maize cysteine protease, the 82E4 promoter, and the promoter of the SAG genes. Suitable anther-specific promoters can be used. Suitable root-preferred promoters known to those skilled in the art can be selected. Suitable promoters for expression preferably in seeds include both seed-specific promoters (promoters active during seed development, such as seed storage polypeptide promoters) and germinating seed promoters (promoters active during seed germination). Such seed-preferred promoters include Cim1 (cytokinin-induced signal); cZ19B1 (19 kDa maize zein); milps (myoinositol-1-phosphate synthase); mZE40-2, also known as Zm-40; nuclc; and celA (cellulose synthase). The gamma-zein gene promoter is an endosperm-specific promoter. The Glob-1 gene promoter is a germ-specific promoter. For dicotyledonous plants, seed-specific promoters include the promoter of the bean bean phaseolin gene, the napin gene, the β-conglycinin gene, the soybean lectin gene, the cruciferin gene, and the like. For monocots, seed-specific promoters include the 15 kD maize zein gene promoter, the 22 kD zein gene promoter, the 27 kD zein gene promoter, the g-zein gene promoter, the molecular weight gamma-zein gene promoter 27 kDa (such as gzw64A promoter, see Genbank accession number S78780), waxy gene promoter, shrunken 1 gene promoter, shrunken 2 gene promoter, globulin 1 gene promoter (see Genbank accession number L22344), Itp2 gene promoter, promoter cim1 gene, maize end1 and end2 gene promoters, nuc1 gene promoter, Zm40 gene promoter, eep1 and eep2 gene promoter; lec1 gene promoter, thioredoxin H gene promoter; mlip15 gene promoter, PCNA2 gene promoter, and shrunken-2 gene promoter.

Examples of inducible promoters include those responsive to pathogens, anaerobic conditions, heat, light, drought, low temperature, or high salt concentration. Pathogen-inducible promoters include promoters of pathogenesis-associated polypeptides (PR polypeptides) that are induced after infection with a pathogen (eg, PR polypeptides, SAR polypeptides, beta-1,3-glucanase, chitinase).

In addition to plant promoters, other suitable promoters may be of bacterial origin, such as the octopine synthase gene promoter , the nopaline synthase gene promoter, and other Ti plasmid-derived promoters, or they may be derived from viral promoters (e.g., the cauliflower mosaic virus 35S and 19S RNA promoters). (CaMV), constitutive promoters of tobacco mosaic virus, 19S and 35S promoters of cauliflower mosaic virus (CaMV), or 35S promoter of boletus mosaic virus).

Suitable methods for introducing polynucleotides into plant cells and then inserting them into the plant genome include microinjection ( Biotechniques (1986) 4:320-334), electroporation ( Proc. Natl. Acad. Sci. USA (1986) 83:5602-5606), transformation, Agrobacterium mediated (US5981840 and US5563055), direct gene transfer ( EMBO J. (1984) 3:2717-2722) and ballistic particle acceleration (US4945050, US5879918, US5886244, US5932782; Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed Gamborg and Phillips (Springer-Verlag, Berlin) (1995) and Biotechnology (1988) 6:923-926).

Mutation

Disclosed is a plant or plant cell containing a mutation in one or more of the polynucleotides or polypeptides described herein, wherein said mutation results in modulation of the function or activity of NtSUS or the polypeptide(s) encoded by them.

A method is provided for modulating the level of an NtSUS polypeptide in a (dried) plant or in a (dried) plant material, said method comprising introducing into the genome of said plant one or more mutations that modulate the expression of at least one NtSUS gene, wherein said at least at least one gene is selected from sequences in accordance with the present invention.

Also provided is a method for identifying a plant with modulated levels of reducing sugars, said method comprising screening a polynucleotide sample from a plant of interest for the presence of one or more mutations in sequences in accordance with the present invention, and optionally matching the identified mutation(s) to the mutation(s). ), which(are) known to modulate(modulate) levels of reducing sugars.

Also disclosed is a plant or plant cell that is heterozygous or homozygous for one or more mutations in the NtSUS gene according to the present invention, said mutation resulting in modulated gene expression or function or activity of the NtSUS polypeptide encoded by it.

A number of approaches can be used to combine mutations in a single plant, including sexual crossing. A plant characterized by the presence of one or more favorable mutations in the heterozygous or homozygous state in a gene according to the present invention that modulate the expression of the gene or the function or activity of the polypeptide encoded by such can be crossed with a plant characterized by the presence of one or more favorable mutations in the heterozygous or homozygous state in one or more genes that modulate their expression or the function or activity of the polypeptide encoded by them. In one embodiment, crosses are carried out to introduce one or more favorable mutations in a heterozygous or homozygous state in a gene in accordance with the present invention in the same plant.

The function or activity of one or more polypeptides of the present invention in a plant is increased or decreased if the function or activity is lower or higher than the function or activity of the same polypeptide(s) in a plant that has not been modified to suppress the function or activity of this polypeptide, and which was cultured, harvested and dried using the same protocols.

In some embodiments, the mutation(s) are introduced into a plant or plant cell using a mutagenesis enabling approach, and the introduced mutation is identified or selected using methods known to those skilled in the art, such as Southern blot analysis, DNA sequencing, PCR analysis or phenotypic analysis. Mutations that affect gene expression or that disrupt the function of the encoded polypeptide can be determined using methods well known in the art. Insertional mutations in exons of a gene, as a rule, result in the formation of non-functional mutant variants. Mutations at conserved residues may be particularly effective in suppressing the metabolic function of the encoded polypeptide. For example, it is understood that a mutation in one or more of the highly conserved regions will likely provide an adjustment to the function of the polypeptide, while a mutation outside of these highly conserved regions will likely have little or no effect on the function of the polypeptide. Additionally, a single nucleotide mutation can result in a stop codon, the presence of which will result in a truncated polypeptide and, depending on the degree of truncation, loss of function.

Methods for producing mutant polynucleotides and polypeptides are also disclosed. Any plant of interest, including a plant cell or plant material, can be genetically modified in a variety of ways known to induce mutagenesis, including site-directed mutagenesis, oligonucleotide-directed mutagenesis, chemical compound-induced mutagenesis, ionizing agent-induced mutagenesis. radiation mutagenesis, mutagenesis using modified bases, mutagenesis using DNA gap duplex, double-strand break mutagenesis, mutagenesis using repair-disrupted host lines, mutagenesis by whole gene synthesis, DNA shuffling, and other equivalent methods.

Also disclosed are fragments of polynucleotides and polypeptides. Polynucleotide fragments may encode polypeptide fragments that retain the biological function of the native polypeptide and are therefore involved in the plant's metabolite transport network. Alternatively, polynucleotide fragments that are useful as hybridization probes or PCR primers generally do not encode polypeptide fragments that retain biological function. In addition, fragments of the disclosed polynucleotides include those fragments that can be assembled into recombinant constructs, as discussed herein. Polynucleotide fragments can range from at least about 25 nucleotides, about 50 nucleotides, about 75 nucleotides, about 100 nucleotides, about 150 nucleotides, about 200 nucleotides, about 250 nucleotides, about 300 nucleotides, about 400 nucleotides, about 500 nucleotides, about 600 nucleotides, about 700 nucleotides, about 800 nucleotides, about 900 nucleotides, about 1000 nucleotides, about 1100 nucleotides, about 1200 nucleotides, about 1300 nucleotides, or about 1400 nucleotides, and up to a full-length polynucleotide encoding the polypeptides described herein. Polypeptide fragments can range from at least about 25 amino acids, about 50 amino acids, about 75 amino acids, about 100 amino acids, about 150 amino acids, about 200 amino acids, about 250 amino acids, about 300 amino acids, about 400 amino acids, about 500 amino acids, and to the full length polypeptide described herein. Mutant variants of a polypeptide can be used to generate mutant, non-naturally occurring or transgenic plants (e.g., mutant, non-naturally occurring, transgenic, human-generated, or genetically engineered plants) or plant cells containing one or more mutant polypeptide variants. . Preferably, mutant variants of the polypeptide retain the function of the unmutated polypeptide. The function of a mutant polypeptide variant may be higher, lower, or approximately the same as that of an unmutated polypeptide.

Mutations in the polynucleotides and polypeptides described herein may include human-made mutations, or artificial mutations, or genetically engineered mutations. Mutations in the polynucleotides and polypeptides described herein may be mutations that are made or can be made by a method that includes an in vitro or in vivo manipulation step. Mutations in the polynucleotides and polypeptides described herein may be mutations that are made or that can be made by a method that involves human intervention.

Methods by which a mutation is randomly introduced into a polynucleotide may include chemical mutagenesis and radiation mutagenesis. Chemical mutagenesis involves the use of exogenously added chemicals such as mutagenic, teratogenic, or carcinogenic organic compounds to induce mutations. To create mutations, mutagens can be used that create primarily point mutations and short deletions, insertions, missense mutations, simple repeat sequences, transversions and/or transitions, including chemical mutagens and radiation. Mutagens include ethyl methanesulfonate, methyl methanesulfonate, N-ethyl-N-nitrosourea, triethylmelamine, N-methyl-N-nitrosourea, procarbazine, chlorambucil, cyclophosphamide, diethyl sulfate, acrylamide monomer, melphalan, nitrogen mustard, vincristine, dimethylnitrosamine, N-methyl-N' -nitro-nitrosoguanidine, nitrosoguanidine, 2-aminopurine, 7,12-dimethyl-benz(a)anthracene, ethylene oxide, hexamethylphosphoramide, bisulfan, diepoxyalkanes (diepoxyoctane, diepoxybutane, etc.), 2-methoxy-6-chloro-9 [3-(ethyl-2-chloroethyl)aminopropylamino]acridine dihydrochloride and formaldehyde.

Also contemplated are spontaneous mutations at the locus, which may not be directly caused by the mutagen, provided that they result in the desired phenotype. Suitable mutagenic agents may also include, for example, ionizing radiation such as x-rays, gamma rays, fast neutron radiation and ultraviolet radiation. The dose of the mutagenic chemical or radiation is determined experimentally for each type of plant tissue so as to obtain a mutation rate that is below a threshold level characterized by lethality or reproductive sterility. Any method for obtaining a plant polynucleotide known to those skilled in the art can be used to obtain a plant polynucleotide for mutation screening.

The mutation method may include one or more plant crossing steps.

Once a mutation has been introduced, screening can be performed to identify mutations that give rise to genes with premature stop codons or otherwise non-functional genes. Once a mutation has been introduced, screening can be performed to identify mutations that give rise to functional genes that are capable of being expressed at increased or decreased levels. Screening for mutant variants can be performed by sequencing, or by using one or more probes or primers specific for a given gene or polypeptide. Polynucleotides can also be engineered to generate specific mutations that can result in modulation of gene expression, modulation of mRNA stability, or modulation of polypeptide stability. In this document, such plants are referred to as "non-naturally occurring" or "mutant" plants. Typically, mutant or non-naturally occurring plants contain at least a portion of the foreign or synthetic or human-made nucleotide (eg, DNA or RNA) that was not present in the plant prior to being manipulated. The foreign nucleotide may be one nucleotide, two or more nucleotides, two or more contiguous nucleotides, or two or more non-contiguous nucleotides such as at least 10, 20, 30, 40, 50,100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400 or 1500 or more contiguous or non-contiguous nucleotides.

Transgenics and gene editing

In addition to effecting mutagenesis, compositions that can modulate the expression or function or activity of one or more of the NtSUS polynucleotides or polypeptides encoded by them include sequence-specific polynucleotides that can disrupt the transcription of one or more endogenous gene(s); sequence-specific polynucleotides that can disrupt the translation of RNA transcripts (eg, double-stranded RNA, siRNA, ribozymes); sequence-specific polynucleotides that can disrupt the stability of one or more polypeptides; sequence-specific polynucleotides that can interfere with the enzymatic function of one or more polypeptides or the function of binding one or more polypeptides to substrates or regulatory polypeptides; antibodies that show specificity for one or more polypeptides; small molecule compounds that can interfere with the stability of one or more polypeptides, or the enzymatic function of one or more polypeptides, or the binding function of one or more polypeptides; zinc finger polypeptides that bind one or more polynucleotides; and meganucleases that act on one or more polynucleotides. Gene editing technologies, genetic editing technologies and genome editing technologies are well known in the art.

Zinc finger nucleases

Zinc finger polypeptides can be used to modulate the expression or function or activity of one or more of the NtSUSα polynucleotides described herein. In various embodiments, a genomic DNA sequence containing part or all of the coding sequence of a polynucleotide is modified by zinc finger nuclease-mediated mutagenesis. In the genomic DNA sequence, a search is made for a unique site for binding the polypeptide to zinc fingers. Alternatively, the genomic DNA sequence is searched for two unique zinc finger binding sites for the polypeptide, with both sites on opposite strands and close to each other, e.g. 1, 2, 3, 4, 5, 6 apart. or more base pairs apart. Accordingly, zinc finger polypeptides are provided that bind to polynucleotides.

A zinc finger polypeptide can be designed to recognize a selected target site in a gene. A zinc finger polypeptide may comprise any combination of motifs derived from natural zinc finger DNA binding domains and non-natural zinc finger DNA binding domains by truncation or extension or a site-directed mutagenesis technique in combination with by a selection method such as, but not limited to, phage display selection, bacterial two-hybrid system selection, or bacterial single-hybrid system selection. The term "non-natural zinc finger DNA binding domain" refers to a zinc finger DNA binding domain that binds a three base pair sequence in a target polynucleotide and that does not occur in a cell or organism containing the polynucleotide to be modifications. Methods for designing a zinc finger polypeptide that binds specific polynucleotides that are unique to a target gene are known in the art.

In other embodiments, a zinc finger polypeptide can be selected for binding to a regulatory sequence of a polynucleotide. More specifically, the regulatory sequence may contain a transcription initiation site, a start codon, an exon region, an exon-intron interface, a terminator, or a stop codon. Accordingly, the present invention provides mutant, non-naturally occurring, or transgenic plant or plant cells obtained by zinc finger nuclease-mediated mutagenesis near or within one or more of the polynucleotides described herein, and methods for producing such plant or plant cells by zinc finger nuclease mediated mutagenesis. The methods for delivering the zinc finger polypeptide and the zinc finger nuclease to the plant are similar to those described below for delivery of the meganuclease.

Meganucleases

In another aspect, methods are described for producing mutant, non-naturally occurring, or transgenic, or otherwise genetically modified plants using meganucleases such as I-CreI. Naturally occurring meganucleases, as well as recombinant meganucleases, can be used to specifically double-strand break at a single site, or at a relatively small number of sites, in a plant's genomic DNA to effect degradation of one or more of the NtSUSα polynucleotides described herein. The meganuclease may be an engineered meganuclease with adjusted DNA recognition properties. Meganuclease polypeptides can be delivered to plant cells by a number of different mechanisms known in the art.

The present invention encompasses the use of meganucleases to inactivate the NtSUSα polynucleotide(s) described herein (or any combination thereof described herein) in a plant cell or plant. In particular, the present invention provides a method for inactivating an NtSUS polynucleotide in a plant using a meganuclease, comprising: (a) providing a plant cell containing an NtSUS polynucleotide described herein; (b) introducing the meganuclease or construct encoding the meganuclease into said plant cell; and (c) allowing the meganuclease to substantially inactivate the NtSUS polynucleotide(s).

Meganucleases can be used to cleave at meganuclease recognition sites within the coding regions of a polynucleotide. Such cleavage often results in deletion of the DNA at the meganuclease recognition site, followed by repair of the mutagenic DNA by joining the non-homologous ends. Such mutations in the coding sequence of a gene are usually sufficient to inactivate the gene. This method for modifying a plant cell comprises, firstly, delivering a meganuclease expression cassette into the plant cell using a suitable transformation method. To achieve maximum efficiency, it is necessary to link the meganuclease expression cassette to a selectable marker and select successfully transformed cells in the presence of a selective agent. This approach will result in the integration of the meganuclease expression cassette into the genome, which, however, may be undesirable if the plant may require authorization. In such cases, the meganuclease expression cassette (and associated selectable marker gene) can be segregated in subsequent generations of the plant using conventional breeding techniques.

Following delivery of the meganuclease expression cassette, the plant cells are grown, initially, under conditions that are typical of the particular transformation procedure used. This may mean growing the transformed cells in a medium below 26°C, often in the dark. Such standard conditions can be applied over a period of time, preferably 1-4 days, to ensure the recovery of the plant cell after the transformation process. At any time after this initial recovery period, the growth temperature can be increased to stimulate the function of the engineered meganuclease to cleave and mutate at the meganuclease recognition site.

TALEN

One way to edit a gene involves the use of effector nucleases like transcription activator (TALEN), which induce the formation of double-strand breaks, to which the cell can respond through repair mechanisms. NHEJ reconnects DNA on both sides of the double strand break where there is very little or no sequence overlap to allow for annealing. This repair mechanism induces errors in the genome through insertion or deletion or chromosomal rearrangement. Any such errors may render the gene products encoded at that location non-functional. Certain applications may require precise removal of the NtSUS polynucleotide from the plant genome. Such applications are possible using a pair of engineered meganucleases, each of which cleaves a meganuclease recognition site on either side of the intended deletion. It is also possible to use TALENs, which are able to recognize and bind to a gene and introduce a double-strand break into the genome. Thus, in another aspect, methods are provided for producing mutant, non-naturally occurring or transgenic or otherwise genetically modified plants described herein using TAL effector nucleases.

CRISPR/Cas

Another method of gene editing involves the use of the bacterial CRISPR/Cas system. Bacteria and archaebacteria are characterized by the presence of chromosomal elements called regularly spaced short palindromic repeats (CRISPR), which are part of the adaptive immune system that protects against viral and plasmid DNA entry. In the case of type II CRISPR systems, CRISPR RNAs (crRNA) function with transactivating crRNA (tracrRNA) and CRISPR-associated (Cas) polypeptides to introduce double-strand breaks into the target DNA. Cleavage of the target by Cas9 requires crRNA and tracrRNA base pairing as well as crRNA and target DNA base pairing. Target recognition is facilitated by the presence of a short motif, called the protospacer-adjacent motif (PAM), which corresponds to the NGG sequence. This system can be used for genome editing. Cas9 is usually programmed with a double RNA consisting of crRNA and tracrRNA. However, the core components of these RNAs can be combined into a single hybrid "guide RNA" to target Cas9. The use of non-coding guide RNA to target DNA for site-specific cleavage promises to be significantly simpler than existing technologies such as TALEN. With the CRISPR/Cas strategy, retargeting of the nuclease complex only requires the introduction of a new RNA sequence, and there is no need to reverse engineer the specificity of the transcription factors based on the polypeptide. CRISPR/Cas technology has been implemented in plants according to the method of WO 2015/189693 A1, which discloses a virus-mediated genome editing platform that is widely applicable to various plant species. Tobacco rattle virus (TRV) RNA2 genome was designed to carry and deliver guide RNA to Nicotiana benthamiana plants overexpressing Cas9 endonuclease. In the context of the present invention, a guide RNA can be derived from any of the NtSUSβ polynucleotide sequences disclosed herein, and the ideas from WO 2015/189693 A1 are used to edit the genome of a plant cell and obtain the desired plant mutant. The rapid pace of technology development has given rise to a huge variety of protocols with wide applicability across the plant kingdom, and these protocols have been well cataloged in a number of recent scientific review articles (e.g., Plant Methods (2016) 12:8; and Front Plant Sci. (2016) 7:506). A review of CRISPR/Cas systems with particular reference to their application in plants is found in Biotechnology Advances (2015) 33, 1, p41-52. Bortesi and Fischer are also making comparisons between CRISPR/Cas technology, zinc finger nucleases and TALEN. Recent developments for the application of CRISPR/Cas in the manipulation of plant genomes are described in Acta Pharmaceutica Sinica B (2017) 7, 3, p292-302) and Curr. Op. in Plant Biol. (2017) 36, 1-8. CRISPR/Cas9 based plasmids for use in plants are listed at "addgene", a non-commercial plasmid repository (addgene.org), and CRISPR/Cas9 based plasmids are commercially available.

Antisense modification

Antisense oligonucleotide technology is another well known method that can be used to modulate the expression of an NtSUS polypeptide. The NtSUS.beta . gene polynucleotide to be repressed is cloned and operably linked to a regulatory region and a transcription termination sequence so that the antisense RNA strand is transcribed. Then, a plant cell is transformed with a recombinant construct and an antisense RNA strand is obtained. The polynucleotide is not necessarily the complete sequence of the gene to be repressed, but is generally substantially complementary to at least a portion of the sense strand of the gene to be repressed.

The polynucleotide can be transcribed into a ribozyme or catalytic RNA that affects the expression of the mRNA. Ribozymes can be designed to specifically pair with virtually any target RNA and cleave the phosphodiester backbone at a specific location, thereby functionally inactivating the target RNA. Heterologous polynucleotides may encode ribozymes designed to cleave specific mRNA transcripts, thus preventing expression of the polypeptide. Hammerhead-type ribozymes are useful for degrading specific mRNAs, although various ribozymes that cleave mRNAs at site-specific recognition sequences can be used. Hammerhead ribozymes cleave the mRNA at sites defined by flanking regions that form complementary base pairs with the target mRNA. The only requirement is that the target RNA must contain a 5'-UG-3' polynucleotide. The construction and production of hammerhead ribozymes is known in the art. Hammerhead ribozyme sequences can be inserted into stable RNA such as transfer RNA (tRNA) to increase the efficiency of in vivo cleavage.

In one embodiment, the sequence-specific polynucleotide that can disrupt translation of the RNA transcript(s) is an interfering RNA. RNA interference or RNA silencing is an evolutionarily conserved process by which specific mRNAs can be targeted for enzymatic degradation. Double-stranded RNA (double-stranded RNA) is introduced into the cell or produced in the cell (for example, double-stranded RNA virus or interfering RNA polynucleotides) to initiate the interfering RNA pathway. Double-stranded RNA can be converted into multiple 21-24 bp small interfering RNA (siRNA) duplexes. using RNase III, which are endonucleases specific for double-stranded RNA. Subsequently, siRNAs can be recognized by RNA-induced silencing complexes that promote siRNA unwinding in an ATP-dependent process. The untwisted siRNA antisense strand directs activated RNA-induced silencing complexes to a targeted mRNA that contains a sequence complementary to the siRNA antisense strand. The targeted mRNA and the antisense strand can form an A-coil, and the major groove of the A-coil can be recognized by activated RNA-induced silencing complexes. The target mRNA can be cleaved by activated RNA-induced silencing complexes at a single site defined by the binding site of the 5'end of the siRNA strand. Activated RNA-induced silencing complexes can be reused to catalyze another cleavage event.

Interfering RNA expression vectors may contain interfering RNA constructs encoding interfering RNA polynucleotides that mediate RNA interference by downregulating mRNA, pre-mRNA, or related RNA variants. Expression vectors may contain an upstream promoter operably linked to an interfering RNA construct, as further described herein. Interfering RNA expression vectors may contain a suitable minimal core promoter, an interfering RNA construct of interest, an upstream (5') regulatory region, a downstream (3') regulatory region, including transcription termination and polyadenylation signals, and other sequences known to those skilled in the art, such as various selectable markers.

Double-stranded RNA molecules may include siRNA molecules assembled from a single oligonucleotide in a stem-loop structure, where the self-complementary sense and antisense portions of the siRNA molecule are connected by polynucleotide-based or non-polynucleotide-based linker(s), as well as circular single-stranded RNA with two or more looped structures and a stem containing self-complementary sense and antisense strands, where the circular RNA can be processed either in vivo or in vitro to form an active siRNA molecule capable of mediating interfering RNA.

The use of small hairpin RNA molecules is also contemplated. They contain a specific antisense sequence in addition to a reverse complementary (sense) sequence, usually separated by a spacer or loop sequence. Cleavage of the spacer or loop provides a single-stranded RNA molecule and its reverse complementary strand so that they can be annealed to form a double-stranded RNA molecule (optionally with additional processing steps that may result in the addition or removal of one, two, three or more nucleotides from the 3' or 5' end of one or both strands). The spacer may be of sufficient length to allow the antisense and sense sequences to anneal and form a double strand structure (or stem) prior to cleavage of the spacer (and optionally subsequent processing steps that may result in the addition or deletion of one, two, three, four or more nucleotides from the 3' end or from the 5' end of one or both strands). The spacer sequence is typically an unrelated polynucleotide that resides between two complementary regions of polynucleotides that, after annealing to a double-stranded polynucleotide, form a small hairpin RNA. The spacer sequence typically contains from about 3 to about 100 nucleotides.

Any RNA polynucleotide of interest can be obtained by selecting an appropriate sequence composition, loop size and stem length to produce a hairpin duplex. When designing, a suitable stem length range for a hairpin duplex includes stem lengths of at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides, such as about 14-30 nucleotides. , approximately 30-50 nucleotides, approximately 50-100 nucleotides, approximately 100-150 nucleotides, approximately 150-200 nucleotides, approximately 200-300 nucleotides, approximately 300-400 nucleotides, approximately 400-500 nucleotides, approximately 500-600 nucleotides, and approximately 600-700 nucleotides. When designing, a suitable range of loop lengths for the hairpin duplex includes a loop length of about 4-25 nucleotides, about 25-50 nucleotides, or more if the stem length of the hairpin duplex is significant. In certain embodiments, the double-stranded RNA or ssRNA molecule is about 15 to about 40 nucleotides in length. In another embodiment, the siRNA molecule is a double-stranded RNA or ssRNA molecule from about 15 to about 35 nucleotides in length. In another embodiment, the siRNA molecule is a double-stranded RNA or ssRNA molecule from about 17 to about 30 nucleotides in length. In another embodiment, the siRNA molecule is a double-stranded RNA or ssRNA molecule from about 19 to about 25 nucleotides in length. In another embodiment, the siRNA molecule is a double-stranded RNA or ssRNA molecule from about 21 to about 23 nucleotides in length. In certain embodiments, hairpin structures with duplex regions longer than 21 nucleotides can facilitate efficient siRNA-driven silencing regardless of loop sequence and length. Exemplary sequences for performing RNAi are described herein.

The target mRNA sequence is typically about 14 to about 50 nucleotides in length. Therefore, the target mRNA can be screened for regions of about 14 to about 50 nucleotides in length that preferably meet one or more of the following criteria: an A+T/G+C ratio of about 2:1 to about 1:2; the presence of an AA dinucleotide or a CA dinucleotide at the 5' end; the presence of a sequence of at least 10 consecutive nucleotides that is unique to the target mRNA (ie, the sequence is not present in other mRNA sequences from the same plant); and the absence of "strings" of more than three consecutive nucleotides representing guanine (G) or more than three consecutive nucleotides representing cytosine (C). Evaluation against these criteria can be performed using various techniques known in the art, for example, computer programs such as BLAST can be used to search public databases to determine if a selected sequence is unique to a target mRNA. Alternatively, the sequence can be selected (and the siRNA sequence designed) using commercially available computer software (eg, OligoEngine, Target Finder and Design Tool for siRNA, which are commercially available).

In one embodiment, target mRNA sequences are selected that are from about 14 to about 30 nucleotides in length and that meet one or more of the criteria above. In another embodiment, sequences are selected that are from about 16 to about 30 nucleotides in length that meet one or more of the criteria above. In a further embodiment, sequences are selected that are from about 19 to about 30 nucleotides in length that meet one or more of the criteria above. In another embodiment, sequences are selected that are from about 19 to about 25 nucleotides in length that meet one or more of the criteria above.

In an exemplary embodiment, the siRNA molecules contain a specific antisense sequence that is complementary to at least 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous nucleotides from any of the polynucleotides described herein.

The specific antisense sequence contained in the siRNA molecule may be identical or substantially identical to the complementary sequence. In one embodiment, the specific antisense sequence contained within the siRNA molecule is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identical. , complementary to the target mRNA sequence. Methods for determining sequence identity are known in the art and can be determined, for example, using the BLASTN program from the University of Wisconsin Computer Group (GCG) software, or provided on the NCBI website.

One method for inducing double-stranded RNA silencing in plants is transformation with a gene construct producing hairpin RNA (see Nature (2000) 407, 319-320). Such constructs contain inverted portions of the target gene sequence separated by an appropriate spacer. Insertion of a functional plant intron region as a spacer fragment further enhances the efficiency of gene silencing induction due to the production of hairpin RNA based on intron splicing ( Plant J. (2001), 27, 581-590). Preferably, the length of the stem is from about 50 nucleotides to about 1 thousand nucleotides in length. Methods for producing hairpin RNA based on intron splicing are well described in the art (see, for example, Bioscience, Biotechnology, and Biochemistry (2008) 72, 2, 615-617).

Interfering RNA molecules having a duplex or double-stranded structure, such as double-stranded RNA or small hairpin RNA, may have blunt ends, or may have 3' or 5' projections. As used herein, the term "overhang" refers to an unpaired nucleotide or nucleotides that protrude from the structure as a duplex if the 3' end of one strand of RNA extends beyond the 5' end of the other strand (3' overhang), or vice versa ( 5' ledge). Nucleotides constituting the protrusions may be ribonucleotides, deoxyribonucleotides, or modified versions thereof. In one embodiment, at least one strand of the interfering RNA molecule has a 3' overhang of about 1 to about 6 nucleotides in length. In other embodiments, the 3' overhang is about 1 to about 5 nucleotides, about 1 to about 3 nucleotides, and about 2 to about 4 nucleotides in length.

If the interfering RNA molecule contains a 3'-protrusion at one end of the molecule, the other end may be blunt-ended, or also have a protrusion (5' or 3'). If the interfering RNA molecule contains a protrusion at both ends of the molecule, the length of the protrusions may be the same or different. In one embodiment, the interfering RNA molecule contains 3' overhangs of about 1 to about 3 nucleotides at both ends of the molecule. In a further embodiment, the interfering RNA molecule is a double stranded RNA having a 2 nucleotide 3' overhang at both ends of the molecule. In yet another embodiment, the nucleotides constituting the interfering RNA overhang are TT dinucleotides or UU dinucleotides.

Interfering RNA molecules may contain one or more 5' or 3' cap structures. The term "cap structure" refers to a chemical modification included at either end of an oligonucleotide that protects the molecule from degradation by endonucleases and may also facilitate delivery or localization within a cell.

Another modification applied to interfering RNA molecules is the chemical coupling to the interfering RNA molecule of one or more fragments or conjugates that enhance the function, cellular distribution, cellular uptake, bioavailability, or stability of the interfering RNA molecule. Polynucleotides can be synthesized or modified using methods conventional in the art. Chemical modifications include 2' modifications, introduction of non-natural bases, covalent attachment of a ligand, and replacement of phosphate bonds with thiophosphate bonds. In this embodiment, the strength of the duplex structure is strengthened by at least one and typically two chemical bonds.

The nucleotides of one or both of the two single strands can be modified to modulate the activation of cellular enzymes such as, for example, but not limited to, certain nucleases. Techniques for reducing or inhibiting cellular enzyme activation are known in the art and include, but are not limited to, 2'-amino modifications, 2'-fluoro modifications, 2'-alkyl modifications, uncharged backbone modifications, morpholine modifications, 2'-O-methyl modifications, and phosphoramidate.

The interfering RNA molecule can be conjugated to ligands, for example, to enhance its absorption into the cell. In certain embodiments, the hydrophobic ligand is conjugated to a molecule to facilitate direct penetration across the cell membrane. In certain instances, conjugation of the cationic ligand to oligonucleotides often results in improved nuclease resistance.

"Targeted induced local lesions in the genome" (TILLING) is another mutagenesis technology that can be used to create and/or identify polynucleotides encoding polypeptides with modified expression, function and/or activity. TILLING also allows selection of plants carrying such mutant variants. TILLING combines high density mutagenesis with high throughput screening methods. TILLING methods are well known in the art (see McCallum et al ., (2000) Nat Biotechnol 18: 455-457 and Stemple (2004) Nat Rev Genet 5(2): 145-50).

Various embodiments are directed to expression vectors containing one or more of the NtSUS polynucleotides or interfering RNA constructs that contain one or more of the NtSUS polynucleotides described herein.

Various embodiments are directed to expression vectors containing one or more of the NtSUS polynucleotides or one or more interfering RNA constructs described herein.

Various embodiments are directed to expression vectors containing one or more NtSUS polynucleotides or one or more interfering RNA constructs encoding one or more interfering RNA polynucleotides described herein that are capable of self-hybridizing to form a hairpin structure, wherein the construct comprises (a) one or more of the NtSUS polynucleotides described herein; (b) a second sequence encoding a spacer element that forms a loop of the hairpin structure; and (c) a third sequence containing a sequence inversely complementary to the first sequence located in the same orientation as the first sequence, where the second sequence is located between the first sequence and the third sequence, and the second sequence is operably linked to the first sequence and the third sequence.

The disclosed NtSUS polynucleotides can be used to construct various polynucleotides that do not form hairpin structures. For example, a double-stranded RNA can be formed by (1) transcribing a first strand of DNA by operably linking to a first promoter and (2) transcribing a sequence reversely complementary to a DNA fragment of the first strand by operably linking to a second promoter. Each polynucleotide strand can be transcribed from a single expression vector, or from different expression vectors. An RNA duplex having RNA interference properties can be enzymatically converted to siRNA to modulate RNA levels.

Thus, various embodiments are directed to expression vectors containing one or more of the NtSUS polynucleotides or interfering RNA constructs described herein encoding interfering RNA polynucleotides capable of self-hybridization, wherein the construct comprises (a) one or more of the NtSUS polynucleotides, described in this document; and (b) a second sequence containing a sequence that is complementary (eg, inversely complementary) to the first sequence in the same orientation as the first sequence.

Various compositions and methods are provided for modulating endogenous expression levels of one or more of the NtSUS polypeptides described herein (or any combination thereof described herein) by promoting co-suppression of gene expression.

Various compositions and methods are provided for modulating the expression level of an endogenous gene by modulating mRNA translation. A host plant (tobacco) cell can be transformed with an expression vector containing a promoter operably linked to an NtSUSβ polynucleotide located in an antisense orientation with respect to the promoter to allow expression of RNA polynucleotides with a sequence complementary to the mRNA.

Various expression vectors for modulating mRNA translation may contain a promoter operably linked to an NtSUSβ polynucleotide in which the sequence is located in an antisense orientation relative to the promoter. The length of the antisense RNA polynucleotides can vary and can be about 15-20 nucleotides, about 20-30 nucleotides, about 30-50 nucleotides, about 50-75 nucleotides, about 75-100 nucleotides, about 100-150 nucleotides, about 150-200 nucleotides and approximately 200-300 nucleotides.

Mobile genetic elements

Alternatively, targeting for gene inactivation can be accomplished by introducing transposons (eg, IS elements) into the genomes of plants of interest. These transposable genetic elements can be introduced by sexual cross-pollination and insertion mutants can be screened for loss of polypeptide function. The disrupted gene of the parent plant can be introduced into other plants by crossing the parent plant with a plant that has not undergone transposon-induced mutagenesis, such as by sexual cross-pollination. Any standard selection techniques known to those skilled in the art can be used. In one embodiment, one or more genes can be inactivated by insertion of one or more transposons. Mutations can lead to homozygous destruction of one or more genes, to heterozygous destruction of one or more genes, or to a combination of homozygous and heterozygous destruction if more than one gene is destroyed. Suitable transposable elements include retrotransposons, retroposons, and SINE-like elements. Such methods are known to those skilled in the art.

Ribozymes

Alternatively, NtSUS polynucleotides can be targeted for inactivation by introducing ribozymes derived from a number of small circular RNAs that are capable of self-cleavage and replication in plants. These RNAs can replicate either independently (viroid RNA) or with the help of a helper virus (satellite RNA). Examples of suitable RNAs include avocado sunspot viroid-derived and satellite RNAs derived from tobacco ringspot virus, alfalfa temporary streak virus, tobacco velvet blotch virus, Solanum nodiflorum sunspot virus, and subterranean clover spot virus. Various target RNA-specific ribozymes are known to those skilled in the art.

4. Plants

Mutant or non-naturally occurring plants or plant cells may have any combination of one or more mutations in one or more NtSUS genes that results in modulated expression or function or activity of those genes or products of those genes. For example, mutated or non-naturally occurring plants or plant cells may have a single mutation in a single NtSUS gene; multiple mutations in the same NtSUS gene; one mutation in two or more, or three or more, or four or more NtSUS genes; or multiple mutations in two or more, or three or more, or four or more NtSUS genes. As a further example, mutated or non-naturally occurring plants or plant cells may have one or more mutations in a particular portion of the NtSUS gene(s), for example, in the region of the NtSUS gene that encodes the active site of the NtSUS polypeptide or a portion thereof. As a further example, mutated or non-naturally occurring plants or plant cells may have one or more mutations in a region outside one or more of the NtSUS genes, such as the region upstream or downstream of the NtSUS gene it regulates, provided that they modulate the function or expression of the NtSUS gene(s). Upstream elements may include promoters, enhancers, or transcription factors. Some elements, such as enhancers, may be upstream or downstream of the gene they regulate. The element(s) are not necessarily adjacent to the gene it regulates, as some elements have been found to be several thousand base pairs upstream or downstream from the gene they regulate. Mutant or non-naturally occurring plants or plant cells may have one or more mutations located in the first 100 nucleotides from the gene(s), in the first 200 nucleotides from the gene(s), in the first 300 nucleotides from the gene(s), in the first 400 nucleotides from a gene(s), in the first 500 nucleotides from a gene(s), in the first 600 nucleotides from a gene(s), in the first 700 nucleotides from a gene(s), in the first 800 nucleotides from a gene(s), in the first 900 nucleotides from a gene(s), in the first 1000 nucleotides from a gene(s), in the first 1100 nucleotides from a gene(s), in the first 1200 nucleotides from a gene(s), in the first 1300 nucleotides from a gene(s), in the first 1400 nucleotides from the gene(s) or in the first 1500 nucleotides from the gene(s). Mutant or non-naturally occurring plants or plant cells may have one or more mutations located in the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, or fifteenth set of 100 nucleotides of a gene(s) or combinations thereof. Disclosed are mutant or non-naturally occurring plants or plant cells (eg, mutant, non-naturally occurring or transgenic plants or plant cells, and the like, as described herein) containing variants of the mutant polypeptide.

In one embodiment, plant seeds are subjected to mutagenesis and then grown into first generation mutant plants. The first generation plants are then allowed to self-pollinate and the seeds from the first generation plants are grown into second generation plants which are then tested for mutations at their loci. Although mutated plant material can be screened for mutations, the advantage of screening second generation plants is that all somatic mutations correspond to mutations in the germline. One of skill in the art will appreciate that various plant material, including, without limitation, seeds, pollen, plant tissue, or plant cells, can be mutated to create mutant plants. However, the type of plant material subjected to mutagenesis may be of importance when a plant polynucleotide is screened for mutations. For example, if pollen is subjected to mutagenesis prior to pollination of an unmutated plant, first generation plants are grown from the seeds obtained from this pollination. Each cell of the first generation plants will contain mutations that originated in the pollen; thus, these first generation plants can then be screened for mutations instead of waiting for the second generation to appear.

Obtaining modified plants, screening and crossing

NtSUSα polynucleotides derived from single plants, plant cells, or plant material can optionally be combined to expedite screening for mutations in a plant population derived from mutated plant tissue, plant cells, or plant material. One or more next generations of plants, plant cells, or plant material can be tested. The size of the optionally pooled group depends on the sensitivity of the screening method used.

After optionally pooling the samples, they can be analyzed using polynucleotide-specific amplification techniques such as PCR. Any one or more primers or probes specific for a gene or sequences immediately adjacent to a gene can be used to amplify sequences within an optionally pooled sample. Preferably, one or more primers or probes are designed to amplify regions of the locus where beneficial mutations are most likely to occur. Most preferably, the primer is designed to detect mutations in regions of the polynucleotide. Additionally, it would be preferred for the primer(s) and probe(s) to avoid known polymorphic sites to facilitate screening for point mutations. To facilitate identification of amplification products, one or more primers or probes can be labeled using any conventional labeling method. Primer(s) or probe(s) can be designed based on the sequences described herein using methods that are well known in the art.

To facilitate identification of amplification products, the primer(s) or probe(s) may be labeled using any conventional labeling method. They can be designed based on the sequences described herein using methods that are well known in the art.

Polymorphisms can be identified by means known in the art and some of those described in the literature.

In some embodiments, a plant can be regenerated or grown from a plant, plant tissue, or plant cell. Any suitable methods for regenerating or growing a plant from a plant cell or plant tissue can be used, such as, without limitation, tissue culture or regeneration from protoplasts. Preferably, plants can be regenerated by growing the transformed plant cells on callus induction medium, shoot induction medium and/or root induction medium. See, for example, McCormick et al., Plant Cell Reports 5:81-84 (1986). These plants can then be grown and either pollinated with the same transformed line or other lines and the resulting hybrid identified as expressing the desired phenotypic characteristic. You can grow two or more generations to ensure that the expression of the desired phenotypic characteristic is stably maintained and inherited, and then collect seeds to ensure that the expression of the desired phenotypic characteristic has been achieved. Thus, the expression "transformed seeds" as used herein refers to seeds that contain a nucleotide construct stably integrated into the plant genome.

Accordingly, in a further aspect, a method for producing a mutant plant is provided. The method includes obtaining at least one plant cell containing the NtSUS gene encoding a functional NtSUS polynucleotide described herein (or any combination thereof described herein). This at least one plant cell is then treated under conditions effective to modulate the function of the NtSUSα polynucleotide(s) described herein. At least one mutant plant cell is then propagated to produce a mutant plant, wherein the mutant plant has modulated levels of the described polypeptide(s) (or any combination thereof described herein) compared to a control plant. In one embodiment of this method for producing a mutant plant, the step of treating includes exposing at least one cell to a chemical mutagenic agent as described above and under conditions effective to produce at least one mutant plant cell. In another embodiment of this method, the treatment step comprises exposing at least one cell to a source of ionizing radiation under conditions effective to produce at least one mutant plant cell. The term "mutant plant" includes mutant plants in which the genotype is modified from that of the control plant, preferably by methods other than genetic engineering and genetic modification.

In certain embodiments, the mutant plant, mutant plant cell, or mutant plant material may contain one or more mutations that occur naturally in another plant, plant cell, or plant material and provide the desired trait. The mutation can be inserted (eg, by introgression) into another plant, plant cell, or plant material (eg, a plant, plant cell, or plant material with a different genetic background from that of the plant from which the mutation occurs) to confer the trait in them. . Thus, as an example, a mutation that occurs naturally in the first plant, can be introduced into a second plant, such as a second plant with a different genetic background from that of the first plant. Thus, a person skilled in the art can search for and identify a plant that naturally carries in its genome one or more mutant alleles of the genes described herein that provide the desired trait. The naturally occurring mutant allele(s) can be transferred to a second plant in a variety of ways, including selection, backcrossing, and introgression to produce lines, varieties, or hybrids that have one or more of the mutations in the genes described herein. The same technique can also be applied to introgression of one or more unnatural mutation(s) from a first plant to a second plant. Plants displaying the desired trait can be selected from a pool of mutant plants. Preferably, selection is made using the polynucleotide data described herein. Therefore, it is possible to carry out selection on a genetic basis in comparison with the control. Such a screening approach may include the use of conventional amplification and/or hybridization techniques as discussed herein. Thus, a further aspect of the present invention relates to a method for identifying a mutant plant, comprising the steps of: (a) obtaining a sample containing a polynucleotideNtSUS from a plant; and (b) determining the sequence of the polynucleotideNtSUS, where the difference is in the polynucleotide sequenceNtSUS compared to polynucleotideNtSUS from a control plant indicates that said plant is a mutant plant. In another aspect, a method is provided for identifying a mutant plant that accumulates reducing sugar(s) at elevated or reduced levels compared to a control plant, comprising the steps of: (a) obtaining a sample from the plant to be screened; (b) determining whether said sample contains one or more mutations in one or more of the polynucleotidesNtSUSdescribed in this document; and (c) determining the level of at least one reducing sugar in said plant. Preferably, the level of at least one reducing sugar is determined in green leaves, leaves subjected to early stages of drying, or leaves subjected to drying. In another aspect, a method is provided for obtaining a mutant plant that is characterized by increased or decreased levels of at least one reducing sugar compared to a control plant, comprising the steps of: (a) obtaining a sample from the first plant; (b) determining whether said sample contains one or more mutations in one or more polynucleotidesNtSUSdescribed in this document, which lead to modulated levels of at least one reducing sugar; and (c) transferring one or more mutations to a second plant. Preferably, the level of at least one reducing sugar is determined in green leaves, leaves subjected to early stages of drying, or leaves subjected to drying. The mutation(s) can be transferred to the second plant by various methods known in the art, such as genetic engineering, gene manipulation, introgression, plant breeding, backcrossing, and the like. In one embodiment, the first plant is a naturally occurring plant. In one embodiment, the second plant has a different genetic background than the first plant. In another aspect, a method is provided for obtaining a mutant plant that is characterized by increased or decreased levels of at least one reducing sugar compared to a control plant, comprising the steps of: (a) obtaining a sample from the first plant; (b) determining whether said sample contains one or more mutations in one or more of the polynucleotidesNtSUSdescribed in this document, which lead to modulated levels of at least one reducing sugar; and (c) introgression of one or more mutations into a second plant. Preferably, the level of at least one reducing sugar is determined in green leaves, leaves subjected to early stages of drying, or leaves subjected to drying. In one embodiment, the introgression step includes plant breeding, optionally including backcrossing and the like. In one embodiment, the first plant is a naturally occurring plant. In one embodiment, the second plant has a different genetic background than the first plant. In one embodiment, the first plant is not a cultivar or elite cultivar. In one embodiment, the second plant is a cultivar or elite cultivar. An additional aspect relates to a mutant plant (including a mutant plant that is a cultivar or elite cultivar) obtained or obtained using the methods described herein. In certain embodiments, "mutant plants" may have one or more mutations located only in a particular region of the plant, for example, within the sequence of one or more polynucleotides.NtSUSdescribed in this document. In this embodiment, the rest of the genomic sequence of the mutant plant will be the same or substantially the same as the plant prior to mutagenesis.

In certain embodiments, mutant plants may have one or more mutations located in more than one region of the plant genome, for example, within the sequence of one or more of the NtSUSβ polynucleotides described herein and in one or more additional regions of the genome. In this embodiment, the rest of the genomic sequence of the mutant plant will not be the same or substantially the same as the plant prior to mutagenesis. In certain embodiments, mutant plants may lack one or more mutations in one or more, two or more, three or more, four or more, or five or more exons of the NtSUSα polynucleotide(s) described herein; or may not have one or more mutations in one or more, two or more, three or more, four or more, or five or more introns of the NtSUS polynucleotide(s) described herein; or may not have one or more mutations in the promoter of the NtSUS polynucleotide(s) described herein; or may lack one or more mutations in the 3'-untranslated region of the NtSUS polynucleotide(s) described herein; or may lack one or more mutations in the 5'-untranslated region of the NtSUS polynucleotide(s) described herein; or may not have one or more mutations in the coding region of the NtSUS polynucleotide(s) described herein; or may lack one or more mutations in the non-coding region of the NtSUS polynucleotide(s) described herein; or any combination of two or more, three or more, four or more, five or more, or six or more parts thereof.

In a further aspect, a method for identifying a plant, plant cell, or plant material containing a mutation in a gene encoding an NtSUSα polynucleotide described herein is provided, comprising: (a) performing mutagenesis of the plant, plant cell, or plant material; (b) obtaining a sample from said plant, plant cell or plant material or their descendants; and (c) determining the polynucleotide sequence of the NtSUS gene or variant or fragment thereof, where a difference in said sequence is indicative of one or more mutations therein. This method also provides for the selection of plants having a mutation(s) that occurs(occurs) in regions of the genome that affect the expression of the NtSUS gene in the plant cell, such as the site of transcription initiation, start codon, intron region, exon border. intron, terminator, or stop codon.

Plant families, species, varieties, seeds and tissue culture

Plants suitable for genetic modification to be applied to them include monocot and dicot plants and plant cell systems, including species from one of the following families: Acanthaceae, Alliaceae, Alstroemeriaceae, Amaryllidaceae, Apocynaceae, Arecaceae, Asteraceae, Berberidaceae, Bixaceae, Brassicaceae , Bromeliaceae, Cannabaceae, Caryophyllaceae, Cephalotaxaceae, Chenopodiaceae, Colchicaceae, Cucurbitaceae, Dioscoreaceae, Ephedraceae, Erythroxylaceae, Euphorbiaceae, Fabaceae, Lamiaceae, Linaceae, Lycopodiaceae, Malvaceae, Melanthiaceae, Musaceae, Myrtaceae, Nyssaceae, Papaveraceae, Pinaceae, Plantaginaceae, Poaceae, Rosaceae , Rubiaceae, Salicaceae, Sapindaceae, Solanaceae, Taxaceae, Theaceae or Vitaceae.

Suitable species may include members of the genus Abelmoschus, Abies, Acer, Agrostis, Allium, Alstroemeria, Ananas, Andrographis, Andropogon, Artemisia, Arundo, Atropa, Berberis, Beta, Bixa, Brassica, Calendula, Camellia, Camptotheca, Cannabis, Capsicum, Carthamus, Catharanthus, Cephalotaxus, Chrysanthemum, Cinchona, Citrullus, Coffea, Colchicum, Coleus, Cucumis, Cucurbita, Cynodon, Datura, Dianthus, Digitalis, Dioscorea, Elaeis, Ephedra, Erianthus, Erythroxylum, Eucalyptus, Festuca, Fragaria, Galanthus, Glycine, Gossypium, Helianthus, Hevea, Hordeum, Hyoscyamus, Jatropha, Lactuca, Linum, Lolium, Lupinus, Lycopersicon, Lycopodium, Manihot, Medicago, Mentha, Miscanthus, Musa, Nicotiana, Oryza, Panicum, Papaver, Parthenium, Pennisetum, Petunia, Phalaris, Phleum, Pinus, Poa, Poinsettia, Populus, Rauwolfia, Ricinus, Rosa, Saccharum, Salix, Sanguinaria, Scopolia, Secale, Solanum, Sorghum, Spartina, Spinacea, Tanacetum, Taxus, Theobroma, Triticosecale, Triticum, Uniola, Veratrum, Vinca, Vitis and Zea.

Suitable species may include Panicum spp., Sorghum spp., Miscanthus spp., Saccharum spp., Erianthus spp., Populus spp., Andropogon gerardii (Gerard's bearded vulture), Pennisetum purpureum (elephantgrass), Phalaris arundinacea (reed grass), Cynodon dactylon (piggy finger), Festuca arundinacea (cane fescue), Spartina pectinata (comb spartina), Medicago sativa (alfalfa), Arundo donax (cane arundo), Secale cereale (rye), Salix spp. (willow), Eucalyptus spp. (eucalyptus), Triticosecale (triticale), bamboo, Helianthus annuus (sunflower), Carthamus tinctorius (safflower), Jatropha curcas (jatropha), Ricinus communis (castor bean), Elaeis guineensis (oil palm), Linum usitatissimum (flax), Brassica juncea, Beta vulgaris (sugar beet), Manihot esculenta (cassava), Lycopersicon esculentum (tomato), Lactuca sativa (lettuce), Musyclise alca (banana), Solanum tuberosum (potato), Brassica oleracea (broccoli, cauliflower, Brussels sprouts) , Camellia sinensis (tea), Fragaria ananassa (strawberry), Theobroma cacao (cocoa), Coffeycliseca (coffee), Vitis vinifera (grape), Ananas comosus (pineapple), Capsicum annum (hot and sweet pepper), Allium cepa (onion) , Cucumis melo (melon), Cucumis sativus (cucumber), Cucurbita maxima (giant squash), Cucurbita moschata (nutmeg squash), Spinacea oleracea (spinach), Citrullus lanatus (watermelon), Abelmoschus esculentus (okra), Solanum melongena (eggplant) , Rosa spp. (rose), Dianthus caryophyllus (carnation), Petunia spp. (petunia), Poinsettia pulcherrima (poinsettia), Lupinus albus (lupine), Uniola paniculata (oats), bent grass (Agrostis spp.), Populus tremuloides (aspen poplar), Pinus spp. (pine), Abies spp. (fir), Acer spp. (maple), Hordeum vulgare (barley), Poa pratensis (bluegrass), Lolium spp. (chaff) and Phleum pratense (timothy), Panicum virgatum (millet), Sorghuycliseor (sorghum, Sudan grass), Miscanthus giganteus (miscanthus), Saccharum sp. (sugarcane), Populus balsamifera (poplar), Zea mays (corn), Glycine max (soybean), Brassica napus (canola), Triticum aestivum (wheat), Gossypium hirsutum (cotton), Oryza sativa (rice), Helianthus annuus ( sunflower), Medicago sativa (alfalfa), Beta vulgaris (sugar beet) or Pennisetum glaucum (pearl millet).

Various embodiments are directed to mutant tobacco, non-naturally occurring tobacco, or transgenic tobacco plants or plant cells modified to modulate gene expression levels, resulting in plants or plant cells, such as a tobacco plant or plant cell, in which the expression level of the polypeptide is modulated. in tissues of interest compared to controls. The disclosed compositions and methods can be applied to any species of the genus Nicotiana , including N. rustica and N. tabacum (eg, LA B21, LN KY171, TI 1406, Basma, Galpao, Perique, Beinhart 1000-1 and Petico). Other species include N. acaulis , N. acuminata, N. africana, N. alata, N. ameghinoi, N. amplexicaulis, N. arentsii, N. attenuata, N. azambujae, N. benavidesii, N. benthamiana, N. bigelovii , N. bonariensis, N. cavicola, N. clevelandii, N. cordifolia, N. corymbosa, N. debneyi, N. excelsior, N. forgetiana, N. fragrans, N. glauca, N. glutinosa, N. goodspeedii, N gossei, N. hybrid, N. ingulba, N. kawakamii, N. knightiana, N. langsdorffii, N. linearis, N. longiflora, N. maritima, N. megalosiphon, N. miersii, N. noctiflora, N. nudicaulis , N. obtusifolia, N. occidentalis, N. occidentalis subsp. hesperis, N. otophora, N. paniculata, N. pauciflora, N. petunioides, N. plumbaginifolia, N. quadrivalvis, N. raimondii, N. repanda, N. rosulata, N. rosulata subsp. ingulba, N. rotundifolia, N. setchellii, N. simulans, N. solanifolia, N. spegazzinii, N. stocktonii, N. suaveolens, N. sylvestris, N. thyrsiflora, N. tomentosa, N. tomentosiformis, N. trigonophylla, N. umbratica, N. undulata, N. velutina, N. wigandioides, and N. x sanderae. In one embodiment, the plant is N. tabacum.

The use of tobacco varieties and elite varieties of tobacco is also contemplated in this document. A transgenic, non-naturally occurring or mutant plant, therefore, may be a variety of tobacco or an elite variety of tobacco that contains one or more transgenes or one or more genetic mutations, or a combination thereof. The genetic mutation(s) (e.g., one or more polymorphisms) may be mutations that do not naturally occur in a particular tobacco variety or tobacco variety (e.g., premium tobacco), or may be a genetic mutation(s) that ( which) exists(exists) in nature, provided that the mutation does not exist naturally in a particular variety of tobacco or variety of tobacco (for example, in an elite variety of tobacco).

Particularly useful varieties of Nicotiana tabacum include Burley-type tobacco, dark-type tobacco, fire-cured tobacco, and oriental-type tobacco. Non-limiting examples of varieties or varieties are: BD 64, CC 101, CC 200, CC 27, CC 301, CC 400, CC 500, CC 600, CC 700, CC 800, CC 900, Coker 176, Coker 319, Coker 371 Gold, Coker 48, CD 263, DF911, DT 538 LC tobacco Galpao, GL 26H, GL 350, GL 600, GL 737, GL 939, GL 973, HB 04P, HB 04P LC, HB3307PLC, hybrid 403LC, hybrid 404LC, hybrid 501 LC , K 149, K 326, K 346, K 358, K394, K 399, K 730, KDH 959, KT 200, KT204LC, KY10, KY14, KY 160, KY 17, KY 171, KY 907, KY907LC, KY14xL8 LC, Little Crittenden, McNair 373, McNair 944, msKY 14xL8, Narrow Leaf Madole, Narrow Leaf Madole LC, NBH 98, N-126, N-777LC, N-7371LC, NC 100, NC 102, NC 2000, NC 291, NC 297 , NC 299, NC 3, NC 4, NC 5, NC 6, NC7, NC 606, NC 71, NC 72, NC 810, NC BH 129 NC 2002 LC, PD 7312 LC, Perique Tobacco, PVH03, PVH09, PVH19, PVH50, PVH51, R 610, R 630, R 7-11, R 7-12, RG 17, RG 81, RG H51, RGH 4, RGH 51, RS 1410, Speight 168, Speight 172, Speight 179, Speight 210, Speight 220, Speight 225, Speight 227, Speight 234, Speight G-28, Speight G-70, Speight H-6, Speight H20, Speight NF3, TI 1406, TI 1269, TN 86, TN86LC, TN 90, TN 97, TN97LC, TN D94, TN D950, TR (Tom Rosson) Madole, VA 309, VA359, AA 37-1, B13P, Xanthi (Mitchell-Mor) , Bel-W3, 79-615, Samsun Holmes NN, KTRDC number 2 hybrid 49, Burley 21, KY8959, KY9, MD 609, PG01, PG04, PO1, PO2, PO3, RG11, RG 8, VA509, AS44, Banket A1 , Basma Drama B84/31, Basma I Zichna ZP4/B, Basma Xanthi BX 2A, Batek, Besuki Jember, C104, Coker 347, Criollo Misionero, Delcrest, Djebel 81, DVH 405, Galpão Comum, HB04P, Hicks Broadleaf, Kabakulak Elassona , Kutsage E1, LA BU 21, NC 2326, NC 297, PVH 2110, Red Russian, Samsun, Saplak, Simmaba, Talgar 28, Wislica, Yayaldag, Prilep HC-72, Prilep P23, Prilep PB 156/1, Prilep P12- 2/1, Yaka JK-48, Yaka JB 125/3, TI-1068, KDH-960, TI-1070, TW136, Basma, TKF 4028, L8, TKF 2002, GR141, Basma xanthi, GR149, GR153, Petit Havana . Sub-varieties of the above with low nicotine to nornicotine conversion are also contemplated, even though they are not specifically mentioned herein.

Embodiments are also directed to compositions and methods for producing mutant plants, non-naturally occurring plants, hybrid plants, or transgenic plants that have been modified to modulate the expression or function of the NtSUS polynucleotide(s) described herein (or any combination thereof). described in this document). Advantageously, the resulting mutant plants, non-naturally occurring plants, hybrid plants or transgenic plants may be similar in general appearance to control plants or substantially the same. Various phenotypic characteristics such as degree of maturity, number of leaves per plant, stem height, leaf ingrowth angle, leaf size (width and length), internode spacing, and lamina-vein ratio can be assessed by field observation.

One aspect relates to the seed of a mutant plant, a non-naturally occurring plant, a hybrid plant, or a transgenic plant described herein. Preferably, the seed is tobacco seed. An additional aspect relates to the pollen or ovule of a mutant plant, non-naturally occurring plant, hybrid plant or transgenic plant described herein. Furthermore, a mutant plant, a non-naturally occurring plant, a hybrid plant, or a transgenic plant as described herein is provided, which further comprises a male sterility polynucleotide.

Also provided is tissue culture from regenerated cells of a mutant plant, non-naturally occurring plant, hybrid plant, or transgenic plant or part thereof, as described herein, wherein plants are regenerated from the culture capable of expressing all of the morphological and physiological characteristics of the parent. Regenerated cells include cells from leaves, pollen, embryos, cotyledons, hypocotyls, roots, root tips, anthers, flowers and parts thereof, ovules, shoots, stems, petioles, pith and bolls, or callus, or protoplasts derived therefrom.

The plant material described herein may be dried tobacco material such as tube-cured tobacco material such as Virginia type tobacco material or sun-cured tobacco material such as Burley and dark type tobacco material. The plant material described herein may be an air-dried tobacco material such as Burley and dark tobacco.

The CORESTA recommendation for tobacco drying is described in CORESTA Handbook No. 17, April 2016, Sustainability in Leaf Tobacco Production.

Modulation of reducing sugars

One goal is to provide mutant, transgenic or non-naturally occurring plants or parts thereof that exhibit modulated levels of at least one reducing sugar in plant material, such as dried leaves. Accordingly, mutant, transgenic or non-naturally occurring plants or parts thereof exhibit modulated levels of at least one reducing sugar compared to a control plant. Preferably, the mutant, transgenic, or non-naturally occurring plants, or parts thereof, have substantially the same appearance as the control plant.

Accordingly, this document describes mutant, transgenic or non-naturally occurring plants or plant parts or cells that are characterized by modulated levels of at least one reducing sugar compared to control cells or control plants. Mutant, transgenic or non-naturally occurring plants or plant cells have been modified to modulate the synthesis or function of one or more of the NtSUS polypeptides described herein by modulating the expression of one or more of the corresponding NtSUS polynucleotides described herein. Accordingly, modulated levels of at least one reducing sugar are observed in at least green leaves, respectively, leaves subjected to early stages of drying, or leaves subjected to drying. In certain embodiments, it is possible to modulate the level of reducing sugars in a plant, for example, in green leaves, preferably leaves subjected to early stages of drying, or dried leaves, or dried tobacco. In certain embodiments, it is possible to modulate the level of reducing sugars in a plant, such as green leaves, preferably early dried leaves, dried leaves, or dried tobacco.

In certain embodiments, the activity and/or expression of one or more of NtSUS1-S, NtSUS1-T, NtSUS2-S, NtSUS2-T, NtSUS3-S, NtSUS3-T, NtSUS4-S, NtSUS4-T, NtSUS5-S, NtSUS5-T, NtSUS6-S and NtSUS6-T.

In certain embodiments, the activity and/or expression of one or more of NtSUS2-S, NtSUS2-T, NtSUS3-S, NtSUS3-T, NtSUS4-S, and NtSUS4-T is modulated.

In certain embodiments, the activity and/or expression of one or more of NtSUS2-S, NtSUS2-T, NtSUS3-S, NtSUS3-T, NtSUS4-S, and NtSUS4-T is modulated and the activity and/or expression of one or more of NtSUS1 is not modulated. -S, NtSUS1-T, NtSUS5-S, NtSUS5-T, NtSUS6-S, and NtSUS6-T.

In certain embodiments, the activity and/or expression of NtSUS2-S, NtSUS2-T, NtSUS3-S, NtSUS3-T, NtSUS4-S, and NtSUS4-T is modulated and the activity and/or expression of NtSUS1-S, NtSUS1-T, NtSUS5 is not modulated -S, NtSUS5-T, NtSUS6-S and NtSUS6-T.

In certain embodiments, the activity and/or expression of one or more of NtSUS2-S, NtSUS3-S, NtSUS3-T, and NtSUS4-S is modulated and the activity and/or expression of one or more of NtSUS1-S, NtSUS1-T, NtSUS2 is not modulated. -T, NtSUS4-T, NtSUS5-S, NtSUS5-T, NtSUS6-S, and NtSUS6-T.

In certain embodiments, the activity and/or expression of NtSUS2-S, NtSUS3-S, NtSUS3-T, and NtSUS4-S is modulated and the activity and/or expression of NtSUS1-S, NtSUS1-T, NtSUS2-T, NtSUS4-T, NtSUS5 is not modulated -S, NtSUS5-T, NtSUS6-S and NtSUS6-T. An additional aspect relates to a mutant, non-naturally occurring or transgenic plant or cell, where the expression or function of one or more of the NtSUS polypeptides described herein is modulated, and the plant part (e.g., green leaves, preferably leaves subjected to early stages of drying, or dried leaves, or dried tobacco) has at least 5% reduced levels of at least one reducing sugar compared to a control plant in which expression or function of said NtSUS polypeptide(s) has not been modulated. In certain embodiments, the level of at least one reducing sugar in a plant, e.g., green leaves, preferably leaves subjected to early stages of drying, or dried leaves, or dried tobacco, can be modulated, for example, by at least about 5% .

Another aspect relates to dried plant material, such as dried leaf or dried tobacco, obtained or derived from mutant, non-naturally occurring or transgenic plants or cells, wherein the expression of one or more of the NtSUS polynucleotides described herein, or the function of the NtSUS polypeptide encoded by them is modulated and wherein the level of at least one reducing sugar is modulated by at least 5% compared to a control plant.

Preferably, the appearance of said plant or part (eg, leaf) is substantially the same as that of a control plant. Preferably, the plant is a tobacco plant or a coffee plant.

Embodiments are also directed to compositions and methods for producing mutant, non-naturally occurring, or transgenic plants or plant cells that have been modified to modulate the expression or function of one or more of the NtSUS polynucleotides or NtSUS polypeptides described herein, which can result in plants or plant components (eg, leaves, such as green leaves, or leaves subjected to early stages of drying, or dried leaves, or tobacco) or plant cells with modulated reducing sugar content.

Mutant, non-naturally occurring or transgenic plants obtained in accordance with the present invention may be similar in appearance to control plants or be essentially the same as control plants. In one embodiment, the leaf weight of the mutant, non-naturally occurring, or transgenic plant is substantially the same as that of the control plant. In one embodiment, the number of leaves in the mutant, non-naturally occurring, or transgenic plant is substantially the same as the control plant. In one embodiment, the leaf weight and number of leaves of the mutant, non-naturally occurring, or transgenic plant is substantially the same as the control plant. In one embodiment, the stem height of the mutant, non-naturally occurring, or transgenic plants is substantially the same as that of control plants at, for example, one, two, or three or more months after being transplanted into the field, or at 10, 20, 30 or 36 or more days after topping. For example, the stem height of the mutant, non-naturally occurring or transgenic plants is no less than the stem height of the control plants. In another embodiment, the chlorophyll content of the mutant, non-naturally occurring, or transgenic plants is substantially the same as that of control plants. In another embodiment, the stem height of the mutant, non-naturally occurring or transgenic plants is substantially the same as the control plants, and the chlorophyll content of the mutant, non-naturally occurring or transgenic plants is substantially the same as the control plants. In other embodiments, the size or shape or number or color of the leaves of the mutant, non-naturally occurring, or transgenic plants are substantially the same as control plants. Preferably, the plant is a tobacco plant or a coffee plant.

In another aspect, a method is provided for modulating the amount of at least one reducing sugar in at least part of a plant (e.g., leaves, such as dried leaves, or in tobacco), comprising the steps of: (i) modulating the expression or function of one or more of the polypeptides The NtSUS described herein (or any combination thereof described herein), preferably, wherein the NtSUS polypeptide(s) are encoded by the corresponding NtSUS polynucleotides described herein; (ii) measuring the level of at least one reducing sugar in at least a portion (e.g., leaves, such as dried leaves, or tobacco, or smoke) of the mutant, non-naturally occurring, or transgenic plant obtained in step (i ); and (iii) identifying a mutant, non-naturally occurring, or transgenic plant in which the level of at least one reducing sugar has been modulated compared to a control plant. Preferably, the appearance of said mutant, non-naturally occurring, or transgenic plant is substantially the same as that of the control plant. Preferably the plant is a tobacco plant.

In another aspect, a method is provided for modulating the amount of at least one reducing sugar in at least a portion of a dried plant material, such as a dried leaf, comprising the steps of: (i) modulating the expression or function of one or more of the NtSUS polypeptides (or any combination thereof). described herein), preferably wherein the NtSUS polypeptide(s) is(are) encoded by the corresponding NtSUS polynucleotides described herein; (ii) collecting plant material, such as one or more leaves, and drying for a specified period of time; (iii) measuring the level of at least one reducing sugar in at least a portion of the dried plant material obtained in step (ii) or during step (ii); and (iv) identifying dried plant material in which the level of at least one reducing sugar has been modulated compared to a control plant.

The increase in expression compared to the control can be from about 5% to about 100%, or the increase is at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or 100% or more, for example, 200%, 300%, 500%, 1000% or more, which includes an increase in transcriptional function, or the expression level of the NtSUS polynucleotide, or the expression level of the NtSUS polypeptide, or a combination thereof.

The increase in function or activity compared to control can be from about 5% to about 100%, or the increase is at least 10%, at least 20%, at least 25%, at least 30%, at least 40 %, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or 100% or more, such as 200%, 300%, 500%, 1000% or more.

The reduction in expression compared to the control may be from about 5% to about 100%, or the reduction is at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or 100% which includes a decrease in transcriptional function, or the expression of an NtSUS polynucleotide, or the expression of an NtSUS polypeptide, or a combination thereof.

The decrease in function or activity compared to control may be from about 5% to about 100%, or the decrease is at least 10%, at least 20%, at least 25%, at least 30%, at least 40 %, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or 100%.

The polynucleotides and recombinant constructs described herein can be used to modulate the expression or function or activity of the NtSUS polynucleotides or NtSUS polypeptides described herein in a plant species of interest, preferably in tobacco.

A variety of polynucleotide-based methods can be used to increase the level of gene expression in plants and plant cells. By way of example, a construct, vector, or expression vector compatible with the plant to be transformed can be prepared that contains the gene of interest together with an upstream promoter capable of causing the gene to be overexpressed in the plant or plant cell. Exemplary promoters are described herein. After transformation and when grown under suitable conditions, the promoter can drive expression to modulate the levels of NtSUS in the plant or in a particular tissue thereof. In one exemplary embodiment, a vector carrying one or more of the NtSUS.beta. polynucleotides described herein (or any combination thereof as described herein) is created to overexpress a gene in a plant or plant cell. The vector carries a suitable promoter, such as the cauliflower mosaic virus CaMV 35S promoter upstream of the transgene, which triggers its constitutive expression in all plant tissues. The vector also carries an antibiotic resistance gene to enable selection of transformed calluses and cell lines.

Expression of sequences from promoters can be enhanced by including expression control sequences, including enhancers, chromatin activating elements, transcription factor responsive elements, and the like. Such control sequences can be constitutive and increase transcription in a versatile manner; or they may be facultative and increase the level of transcription in response to specific signals. Separately, the signals associated with aging and the signals that are active during the drying procedure should be specified.

Thus, various embodiments are directed to methods of modulating the expression level of one or more of the NtSUS polynucleotides described herein (or any combination thereof described herein) by integrating multiple copies of the NtSUS polynucleotide into the plant genome, comprising: transforming a plant cell - host with an expression vector that contains a promoter operably linked to one or more of the NtSUSα polynucleotides described herein. The polypeptide encoded by the recombinant polynucleotide may be a native polypeptide or may be heterologous to the cell.

In one embodiment, the plant for use in the present invention is a plant that has been fire-dried because such plants are high in reducing sugars (greater than about 14% dry weight when grown in the field at the end of drying). Mutant, transgenic, or non-naturally occurring plants or parts thereof that have been fire-dried may have a reducing sugar content that is less than about 14% dry weight when grown in the field at the end of drying, such as less than about 10% dry weight when grown in the field at the end of drying, or less than about 5% dry weight when grown in the field at the end of drying, or less than about 1% dry weight when grown in the field at the end of drying.

In one embodiment, the plant for use in the present invention is a plant that has been sun-dried, since such plants have a reducing sugar content of greater than about 6.8% dry weight when grown in the field at the end of drying. Mutant, transgenic, or non-naturally occurring plants or parts thereof that have been sun-dried may have a reducing sugar content of less than about 5% dry weight when grown in the field at the end of drying, such as less than about 2.5% dry weight. weight when grown in the field at the end of drying, or less than about 1% of the dry weight when grown in the field at the end of drying.

In one embodiment, the plant for use in the present invention is an air-dried plant. Such plants are characterized by a reducing sugar content of greater than about 1.7% dry weight when grown in the field at the end of drying. Mutant, transgenic, or non-naturally occurring plants or parts thereof that have been sun-dried may have a reducing sugar content of less than about 1.5% dry weight when grown in the field at the end of drying, such as less than about 1% dry weight. weight when grown in the field at the end of drying, or less than about 0.5% dry weight when grown in the field at the end of drying.

In certain embodiments, it is preferable to use a fire-dried or sun-dried plant.

Measurement of free amino acids

The content of amino acids can be measured using various methods known from the prior art. One such method is MP 1471 rev 5 2011, Resana, Italy: Chelab Silliker S.r.l, Mérieux NutriSciences Company. In order to detect amino acids in the dried leaves of the plant, after removal of the midrib, the dried lamellae are, if necessary, dried at 40° C. for 2-3 days. The tobacco material is then ground into a fine powder (~100 μM) prior to analysis for amino acid content. Another method for measuring amino acid content in plant material is described in UNI EN ISO 13903:2005. In certain embodiments, free amino acid content is measured in accordance with UNI EN ISO 13903:2005.

Measurement of reducing sugars

The content of reducing sugars can be measured using a segmented flow colorimetric method developed for the analysis of tobacco samples, adapted from Skalar Instrument Co (West Chester, PA) and described in Tobacco Science 20: 139-144 (1976). The measurement of reducing sugars is also described in Coresta Recommended Method 38, CRM38, CRM and ISO 15154: 2003. To determine the reducing sugars in dried leaves, the dried leaflets after removal of the midrib are dried at 40°C for 2-3 days, if necessary. . The tobacco material is then ground to a fine powder (~100 µm) prior to analysis for reducing sugars. In certain embodiments, the measurement of reducing sugars is carried out in accordance with ISO 15154:2003.

Selection

A plant carrying a mutant allele of one or more of the NtSUSβ polynucleotides described herein (or any combination thereof described herein) can be used in a plant breeding program to create useful lines, cultivars and hybrids. In particular, the mutant allele is introgressed into the commercially important varieties described above. Thus, plant breeding methods are provided which involve crossing a mutant plant, a non-naturally occurring plant, or a transgenic plant, as described herein, with a plant having different genetic characteristics. The method may further comprise crossing the progeny plant with another plant, and optionally repeating the crossing until a progeny with the desired genetic traits or genetic background is obtained. One purpose for which such breeding methods are suitable is to introduce the desired genetic trait into other varieties, breeding lines, hybrids or varieties, especially those of commercial interest. Another purpose is to facilitate the accumulation of genetic modifications of various genes in individual varieties, lines, hybrids or plant varieties. Intraspecific as well as interspecific crosses are provided. The progeny plants that result from such crosses, also referred to as breeding lines, are examples of non-naturally occurring plants of the present invention.

In one embodiment, a method for producing a non-naturally occurring plant is provided, comprising: (a) crossing a mutant or transgenic plant with a second plant to produce seed from a progeny tobacco plant; (b) growing the seed of the progeny tobacco plant under conditions suitable for plant growth to produce a non-naturally occurring plant. The method may further comprise: (c) crossing a previous generation of a non-naturally occurring plant with itself or with another plant to produce seed of a progeny tobacco plant; (d) growing the seed of the progeny tobacco plant of step (c) under conditions suitable for plant growth to produce additional non-naturally occurring plants; and (e) repeating the crossing and rearing steps (c) and (d) several times to produce further generations of non-naturally occurring plants. The method may optionally include before step (a) the step of obtaining a parent plant that contains the characterized genetic features and is not identical to the mutant or transgenic plant. In some embodiments, depending on the breeding program, the crossing and rearing steps are repeated 0 to 2 times, 0 to 3 times, 0 to 4 times, 0 to 5 times, 0 to 6 times, 0 to 7 times, 0 to 8 times, 0 to 9 times, or 0 to 10 times to obtain generations of non-naturally occurring plants. Backcrossing is an example of such a method in which a progeny is crossed with one of its parents or with another plant genetically similar to its parent to produce a progeny plant in the next generation that has genetic characteristics more closely related to one of the parents. Plant breeding techniques, in particular plant breeding, are well known and can be used in the methods of the present invention. The present invention further provides non-naturally occurring plants obtained using these methods. Certain embodiments omit the plant selection step.

In some embodiments of the methods described herein, lines resulting from selection and screening for the presence of variant genes are evaluated in the field using standard field procedures. Control genotypes are provided, including the genotype of the original non-mutated parent, and planting sites are located in the field according to a randomized block plan or other appropriate field planning. For tobacco, standard agronomic practices are used, for example, tobacco is harvested, weighed and sampled for chemical and other conventional testing before and during drying. Statistical data analysis is performed to confirm the similarity of the selected lines with the parent line. Cytogenetic analyzes of selected plants are optionally performed to confirm the relationships of chromosome set and chromosome conjugation.

DNA fingerprinting, single nucleotide polymorphism, microsatellite markers, or similar technologies can be used in a marker-assisted selection (MAS) breeding program to transfer or breed mutant gene alleles in other tobacco plants, as described herein. For example, a breeder can create segregated populations by hybridizing a genotype containing a mutant allele with an agronomically desired genotype. Plants from F2 or backcross generations can be screened with a marker derived from the genomic sequence or fragment thereof using one of the techniques listed herein. Plants identified as having the mutant allele can be backcrossed or selfed to create a second population to be screened. Depending on the intended mode of inheritance or the applied MAS technology, it may be necessary for the selected plants to self-pollinate before each backcrossing cycle to facilitate the discovery of the individual plants needed. Backcrossing or other selection procedure can be repeated until the desired phenotype of the recurrent parent is restored.

According to the present invention, in a breeding program, successful crosses produce F1 plants that are fertile. Selected F1 plants can be crossed with one of the parents, and the first generation plants resulting from backcrossing can be self-pollinated to produce a population that is again screened for expression of a variant gene (eg, a null version of the gene). The process of backcrossing, selfing and screening is repeated, for example, at least 4 times until the final screening produces a plant that is fertile and sufficiently similar to the recurrent parent. This plant is self-pollinated, if necessary, and then the progeny are screened again to confirm that the plant is expressing the variant gene. In some embodiments, the F2 generation plant population is screened for expression of the variant gene, e.g., a plant that does not express the polypeptide due to the absence of the gene is identified according to standard methods, e.g., using a PCR technique using primers designed based on the sequence information. polynucleotides for the polynucleotide(s) described herein (or any combination thereof described herein).

Hybrid tobacco varieties can be produced by preventing the female parent plants (ie parental forms) of the first variety from self-pollinating, allowing pollen from the male parental plants of the second variety to fertilize the female parental plants, and producing F1 hybrid seeds on the female plants. Self-pollination of female plants can be prevented by castrating the flowers early in flower development. Alternatively, pollen production on the female parent plants can be prevented using some form of male sterility. For example, male sterility can be achieved through cytoplasmic male sterility (CMS) or transgenic male sterility, where the transgene suppresses microsporogenesis and/or pollen production or causes self-incompatibility. Female parent plants containing CMS are particularly useful. In embodiments in which the female parent plants are characterized by CMS, pollen is collected from the male fertile plants and applied by hand to the stigmas of the CMS female parent plants and the resulting F1 seeds are harvested.

The varieties and lines described herein can be used to form F1 tobacco simple hybrids. In such embodiments, plants of parental varieties can be grown in substantially uniform contiguous populations to facilitate natural cross-pollination of female parent plants by male parent plants. F1 seeds produced on female parent plants are selectively harvested by conventional means. It is also possible to grow two varieties of the parent plant en masse and collect a mixture of F1 hybrid seeds produced on the female and seeds produced on the male by self-pollination. Alternatively, a trilinear cross can be made where a single F1 hybrid is used as the female and crossed with another male. As another alternative, double cross hybrids can be created, where the F1 offspring of two different simple hybrids are crossed with themselves.

A population of mutant, non-naturally occurring, or transgenic plants can be screened or selected for those members of the population that have the desired trait or phenotype. For example, a population from the progeny of a single transformation line can be screened for those plants that have the desired level of expression or function of the polypeptide(s) encoded therewith. Physical and biochemical methods can be used to detect levels of expression or activity. These include Southern analysis or PCR amplification to detect the polynucleotide; Northern blot, RNase S1 protection assay, primer extension assay, or RT-PCR amplification to detect RNA transcripts; enzyme assays to detect the enzymatic or ribozyme function of polypeptides and polynucleotides; and polypeptide gel electrophoresis, Western blotting, immunoprecipitation, and enzyme immunoassays to detect polypeptides. Other techniques such as in situ hybridization, enzyme staining and immunostaining, and enzyme assays can also be used to detect the presence or expression, function or activity of NtSUS polypeptides or NtSUS polynucleotides.

This document describes mutant, non-naturally occurring or transgenic plant and plant cells containing one or more recombinant polynucleotides, one or more polynucleotide constructs, one or more double-stranded RNAs, one or more conjugates, or one or more vectors/expression vectors.

5. Modification of other genes

In particular, the plants and parts thereof described herein can be modified either before or after modulating the expression, function, or activity of one or more NtSUS polynucleotides and/or NtSUS polypeptides in accordance with the present invention.

One or more of the following additional genetic modifications may be present in mutant, non-naturally occurring or transgenic plants or parts thereof.

One or more genes that are involved in the conversion of nitrogen metabolism intermediates can be modified, resulting in lower levels of at least one tobacco-specific nitrosamine (TSNA). Non-limiting examples of such genes include genes encoding nicotine demethylase such as CYP82E4, CYP82E5 and CYP82E10 described in WO2006/091194, WO2008/070274, WO2009/064771 and WO2011/088180, as well as nitrate reductase, described listed in WO2016/046288.

One or more genes that are involved in heavy metal uptake or heavy metal transport may be modified, resulting in lower levels of heavy metals. Non-limiting examples include the polypeptide families associated with multidrug resistance, the cation diffusion mediator (CDF) families, the Zrt-, Irt-like polypeptide (ZIP) family, the cation exchanger (CAX) family, the copper transporter (COPT) family, the heavy metal ATPase family ( e.g. HMA as described in WO2009/074325 and WO2017/129739), the macrophage homologue polypeptide family associated with natural resistance (NRAMP), and other members of the ATP-binding cassette (ABC) transporter family (e.g. MRP) as described in WO2012/028309 which are involved in the transport of heavy metals such as cadmium.

Other illustrative modifications can result in plants with modulated isopropyl malate synthase expression or function, resulting in a change in sucrose ester composition that can be used to change the aroma profile (see WO2013/029799).

Other exemplary modifications can result in plants with modulated threonine synthase expression or function, where methional levels can be modulated (see WO2013/029800).

Other exemplary modifications may result in plants with modulated expression or function of one or more of neoxanthine synthase, lycopene beta cyclase, and 9-epoxycarotenoid dioxygenase to modulate beta-damascenone content to alter flavor profile (see WO2013/064499).

Other exemplary modifications may result in plants with modulated expression or function of chloride channel members of the CLC family to modulate their nitrate levels (see WO2014/096283 and WO2015/197727).

Other exemplary modifications may result in plants with modulated expression or function of one or more asparagine synthetases to modulate asparagine levels in the leaf and provide modulated levels of acrylamide in the aerosol produced by heating or burning the leaf (see WO2017/042162).

Examples of other modifications include modulating herbicide tolerance, for example, glyphosate is an active ingredient in many broad spectrum herbicides. Glyphosate-resistant transgenic plants have been developed by gene transfer of aroA (glyphosate-EPSP synthetase from Salmonella typhimurium and E. coli ). Sulfonylurea-resistant plants were obtained by transformation of the mutant ALS (acetolactate synthetase) gene from Arabidopsis. The photosystem II OB polypeptide from the mutant Amaranthus hybridus was transferred into plants to produce atrazine-resistant transgenic plants; and bromoxynil-resistant transgenic plants were obtained by inserting the bxn gene from the bacterium Klebsiella pneumoniae.

Other examples of modifications result in plants that are resistant to insects. toxinsBacillus thuringiensis(Bt) can provide an efficient path delaying the emergence of Bt-resistant pests, as recently shown on broccoli, where the Bt genescry1Ac And cry1C as part of the "pyramid" provided control of the cabbage moth, resistant to any individual polypeptide, and significantly delayed evolutionary development of resistant insects.

Another exemplary modification results in plants that are resistant to diseases caused by pathogens (eg, viruses, bacteria, fungi). Plants expressing the Xa21 gene (bacterial necrosis resistance) have been developed, with plants expressing both the Bt fusion gene and the chitinase gene (yellow grass moth resistance and rhizoctonia tolerance).

Another exemplary modification results in a fertility adjustment, such as male sterility.

Another exemplary modification results in plants that are tolerant of abiotic stress (eg, drought, temperature change, salinity), and tolerant transgenic plants were obtained by transferring the enzyme acylglycerol phosphate acyltransferase from Arabidopsis; genes encoding mannitol dehydrogenase and sorbitol dehydrogenase, which are involved in the synthesis of mannitol and sorbitol, improve drought tolerance.

Another exemplary modification results in plants in which the activity of one or more endogenous glycosyltransferases, such as N-acetylglucosaminyltransferase, β(1,2)-xylosyltransferase and a(1,3)-fucosyltransferase, is modulated (see WO2011/117249).

Another exemplary modification results in plants in which the activity of one or more nicotine N-demethylases is modulated such that the levels of nornicotine and nornicotine metabolites that form during drying can be modulated (see WO2015169927).

Other exemplary modifications can result in plants with improved storage polypeptide and oil characteristics, plants with increased photosynthetic efficiency, plants with a long shelf life, plants with increased carbohydrate content, and plants resistant to fungi. Also presented are transgenic plants in which the expression of S-adenosyl-L-methionine (SAM) and/or cystathionine gamma synthase (CGS) has been modulated.

One or more genes that are involved in the nicotine synthesis pathway can be modified to produce plants or plant parts that produce modulated levels of nicotine when dried. Nicotine synthesis genes can be selected from the group consisting of: A622, BBLa, BBLb, JRE5L1, JRE5L2, MATE1, MATE 2, MPO1, MPO2, MYC2a, MYC2b , NBB1, nic1, nic2, NUP1, NUP2, PMT1, PMT2, PMT3 , PMT4 and QPT or combinations of one or more of them.

One or more genes that are involved in regulating the amount of one or more alkaloids can be modified to produce plants or plant parts that produce modulated alkaloid levels when dried. Alkaloid control genes may be selected from the group consisting of BBLa , BBLb, JRE5L1, JRE5L2, MATE1, MATE2, MYC2a, MYC2b, nic1, nic2, NUP1, and NUP2 , or a combination of two or more of these.

One or more of these traits can be introgressed into, or directly transformed into, mutant, non-naturally occurring, or transgenic plants of another variety.

In various embodiments, mutant plants, non-naturally occurring plants or transgenic plants, as well as biomass, are provided in which the expression level of one or more polynucleotides of the present invention is modulated to thereby modulate the level of the polypeptide(s) encoded by them.

6. Consumer products

Parts of the plants described herein, in particular the leaf blade and midrib of these plants, can be incorporated into or used in the manufacture of a variety of consumer products, including, but not limited to, aerosol generating materials, aerosol generating devices, smoking articles, smoking articles, smokeless products, medical or cosmetic products, intravenous preparations, tablets, powders and tobacco products. Examples of aerosol forming materials include tobacco compositions, tobacco species, tobacco extract, cut tobacco, cut filler, dried tobacco, exploded tobacco, homogenized tobacco, reconstituted tobacco, and pipe tobacco species. Smoking articles and smoking articles are generally aerosol generating devices. Examples of smoking or smoking articles include cigarettes, cigarillos and cigars. Examples of smokeless products include chewing tobacco and snuff. In certain aerosol generating devices, instead of combustion, the tobacco composition or other aerosol generating material is heated by one or more electrical heating elements to produce an aerosol. In another type of heated aerosol generating device, the aerosol is produced by transferring heat from a combustible fuel cell or heat source to a physically separated aerosol generating material that may be located in, around, or downstream of the heat source. Smokeless tobacco products and various aerosol-forming tobacco-containing materials may contain tobacco in any form, including dried particles, lumps, granules, powders, or a suspension applied to, mixed with, surrounded by, or otherwise combined with other ingredients. in any format such as flakes, films, tablets, foams or pellets. As used herein, the term "smoke" is used to describe the type of aerosol that is produced by smoking articles such as cigarettes or by burning an aerosol-forming material.

In one embodiment, dried plant material is also provided from the mutant, transgenic, and non-naturally occurring plants described herein. Methods for drying green tobacco leaves are known to those skilled in the art and include, without limitation, air drying, fire drying, fire tube drying, and sun drying, as described herein.

In another embodiment, tobacco products are described, including aerosolized tobacco-containing materials containing plant material such as leaves, preferably dried leaves, from mutant tobacco plants, transgenic tobacco plants, or non-naturally occurring tobacco plants described herein. document. The tobacco products described herein may be blended tobacco products, which may additionally contain unmodified tobacco.

7. Products and methods of cultivation and agriculture

For mutant, non-natural or transgenic plants, there may be other uses in, for example, agriculture. For example, the mutant, non-naturally occurring, or transgenic plants described herein can be used to make animal feed and human food.

The present invention also provides methods for producing seeds, comprising cultivating a mutant plant, a non-naturally occurring plant, or a transgenic plant as described herein, and collecting the seeds of the cultivated plants. The seeds of the plants described herein can be conditioned and packaged in a packaging material using means known in the art to form an article of manufacture. Packaging material such as paper or cloth is well known in the art. The seed package may have a label, such as a label or label attached to the packaging material, a label printed on the package, which describes the origin of the seeds it contains.

Compositions, methods, and kits for plant genotyping for identification, selection, or selection may include means for detecting the presence of an NtSUS polynucleotide (or any combination thereof described herein) in a polynucleotide sample. Accordingly, a composition is described comprising one or more primers for specifically amplifying at least a portion of one or more of the NtSUSα polynucleotides, and optionally one or more probes, and optionally one or more amplification or detection reagents.

Accordingly, gene-specific oligonucleotide primers or probes containing about 10 or more contiguous polynucleotides corresponding to the NtSUS polynucleotide(s) described herein are disclosed. Said primers or probes may contain or consist of about 15, 20, 25, 30, 40, 45, or 50 or more contiguous polynucleotides that hybridize (e.g., specifically hybridize) to the NtSUS polynucleotide(s) described herein. . In some embodiments, primers or probes may comprise or consist of about 10-50 contiguous nucleotides, about 10-40 contiguous nucleotides, about 10-30 contiguous nucleotides, or about 15-30 contiguous nucleotides, which can be used in sequence-dependent gene identification methods. (eg, Southern hybridization), or isolation (eg, in situ hybridization of bacterial colonies or bacteriophage plaques), or gene detection (eg, as one or more amplification primers in amplification or detection). One or more specific primers or probes can be designed and used to amplify and detect part or all of a polynucleotide(s). As a specific example, two primers can be used in a PCR protocol to amplify a polynucleotide fragment. PCR can also be performed using one primer that is derived from a polynucleotide sequence and a second primer that hybridizes to a sequence above or below the polynucleotide sequence, such as a promoter sequence, the 3' end of a precursor mRNA, or a sequence derived from a vector. Examples of elevated temperature and isothermal techniques useful for amplifying polynucleotides in vitro are well known in the art. The sample may originate from or be obtained from a plant, plant cell, or plant material, or a tobacco product made from a plant, plant cell, or plant material, as described herein.

In a further aspect, a method is also provided for detecting in a sample the NtSUS polynucleotide(s) described herein (or any combination thereof described herein), comprising the step of: (a) obtaining a sample containing or suspected to contain a polynucleotide; (b) bringing said sample into contact with one or more primers or one or more probes to specifically detect at least a portion of the NtSUS polynucleotide(s); and (c) detecting the presence of an amplification product, wherein the presence of an amplification product is indicative of the presence of the NtSUS polynucleotide(s) in the sample. In a further aspect, the use of one or more primers or probes to specifically detect at least a portion of the NtSUS polynucleotide(s) is also provided. Also provided are kits for detecting at least a portion of an NtSUS polynucleotide(s) that contain one or more primers or probes for specifically detecting at least a portion of an NtSUS polynucleotide(s). The kit may contain polynucleotide amplification reagents, such as PCR reagents, or reagents for probe hybridization detection technology, such as Southern blot, Northern blot, in situ hybridization, or microarray. The kit may contain reagents for antibody binding detection technology such as Western blotting, ELISA, SELDI mass spectrometry, or test strips. The kit may contain reagents for DNA sequencing. The kit may contain reagents and instructions for using the kit.

In some embodiments, the kit may contain instructions for one or more of the methods described. The disclosed kits may be useful for genetic identity determination, phylogenetic studies, genotyping, haplotyping, pedigree analysis or plant breeding, in particular for quantifying codominant traits.

The present invention also provides a method for genotyping a plant, plant cell, or plant material containing an NtSUS.beta. polynucleotide described herein. Genotyping provides a means of distinguishing chromosome pair homologues and can be used to distinguish between segregants in a plant population. Methods based on the use of molecular markers can be used for phylogenetic studies characterizing genetic relationships between crop varieties, identifying hybrids or somatic hybrids, localizing chromosomal segments that affect monogenic traits, cloning based on genetic maps, and studying quantitative inheritance. Any number of molecular marker assays may be used in a particular genotyping method, including those based on amplified fragment length polymorphism (AFLP). AFLP is the result of allelic differences between amplified fragments caused by polynucleotide variability. Thus, the present invention further provides methods for tracking the segregation of one or more genes or polynucleotides, as well as chromosomal sequences genetically related to those genes or polynucleotides, using techniques such as AFLP analysis.

The present invention is further described in the examples below, which are provided to describe the present invention in more detail. These examples, which set out the preferred mode currently provided for the implementation of the present invention, are intended to illustrate and not to limit the present invention.

EXAMPLES

Example 1 Identification of Key Genes for Sucrose Metabolism After Drying in Burley, Virginia and Oriental Tobacco Leaf

To identify key functions contributing to sucrose metabolism during the early leaf drying stages of Burley, Virginia, and Oriental type tobacco, a dominance analysis was performed for the function of genes that are overexpressed in dried leaves after 48 hours of drying compared to mature leaves at time of drying. harvest (log2-fold change >2, adjusted p-value <0.05) of Burley, Virginia and Oriental tobacco. Identified genes involved in the production of reducing sugars and are active after 48 hours of drying, regardless of the types of drying and varieties of tobacco. Tobacco genes involved in the production of reducing sugars have been identified.

The key genes directly involved in the production of reducing sugars during the early stages of drying in leaves belong to the SUS gene family. SUS is likely to be the key enzyme to trigger the accumulation of reducing sugars in the desiccated detached leaves.

The tobacco genome was found to have 12 NtSUS gene products distributed across 6 families, with one S copy and one T copy from each ancestor: NtSUS1-S (SEQ ID NO: 1) , NtSUS1-T (SEQ ID NO: 3) . NtSUS2-S (SEQ ID NO: 5), NtSUS2-T (SEQ ID NO: 7) , NtSUS3-S (SEQ ID NO: 9) , NtSUS3-T (SEQ ID NO: 11) , NtSUS4-S (SEQ ID NO: 13) , NtSUS4-T (SEQ ID NO: 15) , NtSUS5-S (SEQ ID NO: 17) , NtSUS5-T (SEQ ID NO: 19) , NtSUS6-S (SEQ ID NO: 21) and NtSUS6 -T (SEQ ID NO: 23).

The SUS transcripts originated from the genomic sequences NtSUS2-S (SEQ ID NO: 5), NtSUS3-S (SEQ ID NO: 9), NtSUS3-T (SEQ ID NO: 11) and NtSUS4-S (SEQ ID NO: 13). Expression of these genes increased during leaf drying (senescence) as shown in Table 1. This confirms that S copies are particularly involved in the chemical modification of leaves subjected to early drying stages and, in this particular case, in increasing glucose levels and fructose.

Although dried Burley tobacco leaves showed low levels of reducing sugars compared to Virginia and Oriental tobacco (see Figure 1), the NtSUS genes are nevertheless upregulated in Burley tobacco (see Table 1), probably as a constitutive response. to provide, in turn, an available carbon source for amino acid synthesis during the early drying phase.

In both Burley tobacco (BU) and Virginia tobacco (FC), NtSUS1-S and NtSUS1-T , which are not expressed during the early stages of drying (see Table 1), are in particular expressed in the root and stem, indicating about a possible specific function in these tissues, which consists in the delivery of carbohydrates for the synthesis of cell walls or the supply of carbon resources under anoxic conditions. On the other hand , NtSUS3-S, NtSUS3-T, NtSUS4-S , which are induced during the early stages of leaf drying, are also expressed in all organs, while NtSUS2-S and NtSUS2-T are predominantly expressed in immature flowers and petals. NtSUS5-S, NtSUS5-T, NtSUS6-S, and NtSUS6-T are expressed at low levels in all analyzed plant tissues (see Table 2).

Overexpression of NtSUS2-S , NtSUS3-S, NtSUS3-T, and/or NtSUS4-S using an aging-inducible promoter such as SAG12 or E4 could be considered to increase the pool of reducing sugars in desiccated leaves (use of a constitutive promoter can significantly alter plant metabolism). ). On the other hand, knocking out NtSUS2-S , NtSUS3-S, NtSUS3-T and/or NtSUS4-S may contribute to the reduction of reducing sugars in dried leaves.

Example 2 - Expression Silencing NtSUS in Virginia tobacco leaf

NtSUS silencing was investigated to determine if these genes contribute to the reduction of reducing sugars in dried Virginia tobacco leaves. A specific DNA fragment within the coding sequence of both NtSUS was cloned with a strong nocturnal mosaic virus (MMV) promoter into the GATEWAY vector. The NtSUS gene fragment is flanked between MMV and the nos 3'-terminator sequence of the Agrobacterium tumefaciens nopaline synthase gene.

To ensure the selection of plants with low levels of reducing sugars, the leaves of individual T0 plants and corresponding control lines were analyzed after 60 hours of drying to determine the effect on the reduction of reducing sugars. The best T0 lines were selected, showing the lowest level of reducing sugars. Seeds were harvested from these very best T0 lines. T1 progeny were analyzed by qPCR to determine the effectiveness of NtSUS silencing events in reducing the content of reducing sugars.

Manipulating the NtSUS genes (eg, either using a constitutive promoter or a specific senescence-inducible promoter such as SAG12 or E4) can alter the chemistry of dried tobacco leaves. Similarly, knocking out the NtSUS genes using a gene editing strategy such as CRISPR-Cas or mutant selection can alter the amino acid chemistry in the leaves of major commercial tobacco varieties.

Any publication cited or described in this document provides relevant information disclosed prior to the filing date of this application. The statements made in this document should not be construed as an admission that the authors of the present invention have no reason to contrast it with earlier such disclosures. All publications mentioned in the above description are incorporated into this document by reference. Various modifications and variations of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the present invention, as stated, should not be unduly limited to such specific embodiments. Indeed, various modifications of the described methods of carrying out the invention, which are obvious to those skilled in cellular, molecular and plant biology or related fields, are intended to be within the scope of the appended claims.

TABLE 1

Expression of NtSUS genes during the early stages of drying for Burley (BU), Virginia (FC), and Oriental (OR) tobacco

BU FC OR Greens mature 48 hours drying Greens mature 48 hours drying Greens mature 48 hours drying NtSUS1-S 3.4 0.2 0.0 10.4 0.0 0.4 3.4 0.1 0.5 NtSUS1-T 4.1 0.2 0.0 11.4 0.1 0.2 3.4 0.2 0.6 NtSUS2-S 0.2 0.3 28.5 0.3 0.1 1.7 0.6 2.3 17.8 NtSUS2-T 4.0 1.7 5.0 2.6 0.3 0.5 2.7 2.8 3.7 NtSUS3-S 23.4 65.6 130.4 14.2 57.8 82.3 18.7 42.0 163.9 NtSUS3-T 8.5 12.2 76.5 2.4 3.2 41.2 6.1 10.7 126.7 NtSUS4-S 11.6 4.9 22.8 15.5 3.6 22.9 19.0 18.9 105.4 NtSUS4-T 3.2 2.7 8.1 2.7 3.1 1.9 38.0 56.2 37.9 NtSUS5-S 0.6 0.2 0.1 5.1 0.1 0.5 1.4 0.3 0.8 NtSUS5-T 0.5 0.5 0.3 3.6 0.4 0.6 1.1 0.8 1.6 NtSUS6-S 7.8 9.5 8.2 7.6 7.7 5.4 7.8 8.2 8.3 NtSUS6-T 3.1 4.2 4.7 1.6 4.3 3.2 3.7 4.9 5.0

TABLE 2

Expression of NtSUS genes in the root, stem, mid-stem leaf, immature flower (immature flower), sepal, and petal of Burley (BU) and Virginia (FC) tobacco plants grown in the field

BU FC Root Stem Leaf from the middle part of the stem Nezr. flower Sepal Petal Root Stem Leaf from the middle part of the stem Nezr. flower Sepal Petal NtSUS1-S 206.4 294.0 0.9 8.2 1.6 6.4 95.7 129.1 0.7 31.7 1.1 0.7 NtSUS1-T 170.0 348.6 1.0 14.4 2.5 9.0 70.9 137.6 1.1 36.9 1.7 1.7 NtSUS2-S 26.4 17.0 0.4 86.1 12.9 97.9 27.3 13.5 0.4 197.3 7.7 150.3 NtSUS2-T 61.5 55.1 2.2 96.9 37.3 172.7 4.2 12.7 0.4 122.6 12.4 111.4 NtSUS3-S 7.7 23.0 31.8 10.3 22.5 32.4 81.0 98.7 40.4 14.6 67.5 34.6 NtSUS3-T 6.7 6.4 5.0 8.8 7.9 13.7 13.9 20.2 2.8 4.7 6.8 17.7 NtSUS4-S 26.5 40.0 8.5 19.4 14.7 23.4 30.6 43.4 6.6 31.4 10.3 34.1 NtSUS4-T 40.7 29.3 6.4 3.2 6.0 4.1 52.6 47.7 4.8 5.4 12.6 20.4 NtSUS5-S 2.6 6.5 0.5 5.6 1.4 1.1 2.2 2.2 0.3 4.4 0.5 0.4 NtSUS5-T 2.6 5.9 0.6 5.3 1.0 1.9 2.6 2.3 1.2 3.4 0.8 1.8 NtSUS6-S 8.3 7.8 7.8 8.9 6.8 7.1 9.4 8.5 6.6 9.5 7.3 8.6 NtSUS6-T 2.7 2.4 4.1 3.0 3.1 4.9 4.0 3.4 3.6 4.2 3.2 5.2

SEQUENCE LIST

SEQ ID NO: 1: Polynucleotide sequence of NtSUS1-S

atggcagctagtggtcttagcattaagaaaagtttggaggaatccattttggctcatccagatgaaattttggctctcaagtcaaggtacattactacatataatgatattaagaactagaggcttatccaaggttttgttacatttttgaaattataagtttagaacctaatagtacttggtagcacttgtttccttattatctagctgttgttactgcttgttgctactgctttctgttcatctttccttgagcccggtctatcggaaacaacctctctattctcaaagtataaggtttgcgtacatactacctccccagactctacttgtggaatttactgtttttgttgtgttgttgtaatctaatatttattagaattttactgatttttcacatatatatatctatgtcccctgtcgaaaattctatagctcatgttagctaaatacattagtaccattgtttttaattgttttggttttggcacaggattgaaactgaagggaaaggggtaatgaaaccacttgatctcttgaaccatttggtttctgttactagtaagacaaatggagtaaatattgtacctagtgcacttgtggaagttctcagttgcagccaagaagctgtgattgtaccaccaaaactagcactagctgtacgtccgaggcccggtgtatgggagtacttgtcactgaatcttaagacaaagaaagtggctgaattaagcattcctgaataccttcaattgaaagagaacactgttgatgaaaggtaaagtattagtctgcgatttcgctttgtgaaattgaagtttttgttttgattcataatgttttgtgtatcaattatgttaccagtggaaacatattggagttggattttgagccatttacaacagttacaccaccaaaaacactttctgactctattggcaatggtttggagtttcttaatcgccacattgcttcgaaaatgtttcatgataaggagatttccagatgcctccttgacttcctcagaaaccataactacaaaggaaaggtaataaaaaaaagtgtttctttaaacaagttgtatgattatgtgtatatttctaagtatgttaacttgaaaacagtcattgatggtgaaagaaagcattcaaagcctagagagtttccaacttgttctgaaaaaagcagaggaacatttgtgcacattgaatccagaaactccatactccaattttgaatcaaagtttgaagagattggcttggaaagagggtggggaaacaccgctgaacgcgtgcaagacactatcagtcatcttttgcatctccttgaggctcctaacgcgtcttctttggaaaatttccttggtagaatcccattggttttcaatgttgtgattctaactccacatggttattttgctcaagataatgtcttgggctatcctgacactggtggccaggtttgtgtccaatattttgcattcttgatcaagttctttataccatttgaaccaacaatcttnaacattctttttttggttgtgaaatgttgaataggttgtttacattcttgatcaagttccagctatggagcgtgagatgcttcatcgtatgaagcttcaaggactcgatgatatcatccctcgcatccttgttgtaagtggccttaattttcctagtttcatttacacctctaaatgaaattgatcttttttgttgttttatatcaggtaacaaggctgctgcctgatgcagtaggaaccacctgtggcgagcggatggagaaagtatatggggcagaacattctcatataattcgtgttccatttagaactgagaagggaatgttgcgcaaatggatctcacgattcgaagtctggccatacatggaaactttcactgaggttggaacataaaaacaaataaaatccattggaatgttccttctgcaattgaaaatgtcttgctaactgaagacccatttttaaattgatcatcaggatgttgcagaagaacttgtcaaagaattgcaagctaaaccagacttgatcattggaaactacagtgagggaaatcttgctgcctctttgcttgcgaagaaatttggggctactcagtgtactattgctcatgccttggaaaaaactaagtatccaaactctgaccttaattggaagaagtttgatgacaagtatcatttctcaagtcagttcactgctgatctctttgccatgaatcacactgatttcatcatcaccagcactttccaagaaattgctggaaggtaaaagcaaatgcacaccatcatagtatttcatatttttacccttgtttatactatttccattcaccgaccccgacttgtttaggattgagccatagttgttgttgttgtttgtttatactatttccatttgccgaccacaacttgtttaggactgaggtatagttgttgttgttggtttgttcatattattttcattcgctaaccctaacttgtttgggactgaggcatagtagtagtagtagttgttgctattagtttatactatttccatttgccaaccccaacttgtttggtactgagacatagttgttgttgttgttgtttgtttatactatttccatttgccgaccccaacttgtttaggactgaggtatagttgttgttgttggtttgttcatattattttcattcgctaaccccaacttgtttgggactgaggcatagtagtagtagtagtagttgttgctattagtttatactatttccatttgccaaccccaacttgtttggtactgagacatagttgttgttgttgtttgtttatactatttcaatttgtcgaccccaatttgtttgggaccaaggcatggttgttgttgttgtttgtttgtttttactgtttccattgatattggaacatttgttatttgcagcaaaaacactgtaggacagtatgagagtcatactgcttttaccatgcctggattgtaccgagtagtccatggaatcgattcgtttgatccaaagttcaacattgtctcccctggggctgatatgtcaatctacttcccttacactgagaaggagaaaaggctaaccaacttccacccggaaattgaagaactcctctacagtcctgttgagaataaggaccacttgttagtctccttaatttgcttttatttcatcccatttatgatcgcttttatcccaacagatcgattaatcatttgttatcaacataaacagatgtgtgttgaaggaccggaacaagccaattctctttaccatggcaaggctagatcgcgtgaagaatctaacagggctcgtggaatggtatgctaagaatgcaaggctgagggagcttgttaaccttgtggttgtaggcggagacagaaggaaagaatccaaagatttagaagagcaagcagagatgaagaagatgtatgatcttatcgaaacctataacctgaacggccaattcaggtggatttcttcccaaatgaatcgtgtgaggaacggagaactctatcgttacattgcagacacgaggggtgctttcgttcaaccagcattctacgaggcttttggtttgacagttgtagagtctatgacttgtggtttgccaacttttgctacttgtaatggtggaccatttgagattatagtgaatggaaaatctggtttccatattgatcctaatcaaggtgacaaggctgctgatatgttggtaaatttctttgaaaaatctaaagaagatccaagttattgggatgctatttccaagggaggtctgcaacgtattcttgaaaagtaagcttttgcatttgattagcacaagtgcacaaccaagatttaacttttgaacaaactaaaactaacccttttttgtattttcttttgctaggtatacatggcaaatttattcacagaaagtgatcacactatctgggatttatggattctggaagtatgcaaccaagaatgataaagttgctagtgcaaagaagcgctatcttgagatgttttatgaacttggatttaagaaatcagtaagtgtcaattttaaaggggaaccttggatcaacggttaagttgtctttgtgcaacctataggtcaggggtttgagccgtagaagtagccactaatatttacattagggtagactgtgtacatatcacaccccttggggtacggccctttcctggatcctgtatgaacgcgggatgccttgtgcaccgggctgtatttttttttttagtgtcacttctgtattttgtttgagcttgtttataaagtttggaaatctgctgctaatttgtatatttgttggttgtgtatttcaggctgagaaagttccattggctattgatgaatag

SEQ ID NO: 2: NtSUS1-S polypeptide sequence

MAASGLSIKKSLEESILAHPDEILALKSRIETEGKGVMKPLDLLNHLVSVTSKTNGVNIVPSALVEVLSCSQEAVIVPPKLALAVRPRPGVWEYLSLNLKTKKVAELSIPEYLQLKENTVDESGNILELDFEPFTTVTPPKTLSDSIGNGLEFLNRHIASKMFHDKEISRCLLDFLRNHNYKGKSLMVKESIQSLESFQLVLKKAEE HLCTLNPETPYSNFESKFEEIGLERGWGNTAERVQDTISHLLHLLEAPNASSLENFLGRIPLVFNVVILTPHGYFAQDNVLGYPDTGGQVVYILDQVPAMEREMLHRMKLQGLDDIIPRILVVTRLLPDAVGTTCGERMEKVYGAEHSHIIRVPFRTEKGMLRKWISRFEVWPYMETFTEDVAEELVKELQAKPDLIIGNYSEGNLAASLLA KKFGATQCTIAHALEKTKYPNSDLNWKKFDDKYHFSSQFTADLFAMNHTDFIITSTFQEIAGSKNTVGQYESHTAFTMPGLYRVVHGIDSFDPKFNIVSPGADMSIYFPYTEKEKRLTNFHPEIEELLYSPVENKDHLCVLKDRNKPILFTMARLDRVKNLTGLVEWYAKNARLRELVNLVVVGDRRKESKDLEEQAEMK KMYDLIETYNLNGQFRWISSQMNRVRNGELYRYIADTRGAFVQPAFYEAFGLTVVESMTCGLPTFATCNGGPFEIIVNGKSGFHIDPNQGDKAADMLVNFFEKSKEDPSYWDAISKGGLQRILEKYTWQIYSQKVITLSGIYGFWKYATKNDKVASAKKRYLEMFYELGFKKSAEKVPLAIDE

SEQ ID NO: 3: Polynucleotide sequence of NtSUS1-T

atggcaggcagtggtcttagcattaaggaaagtttggaggaatccattttggctcatccagatgaaattttggctcaagtcaaggtacattactgcataatgatattaagacctagaagcggatccaagattttgttacatttttgaaattataagtttagaatctaatatttgttatcgcttgtttccttattatcttgctgttgttactgcctgttgctactagt ttctgttcatccttccttgagctgagtttctatcggaaacaacctctctactctcaaagtaggaataagttatgcgtacacactaccctccccagactccacttgtgtaatttactgagttgttgttgttgttgttgtaatctaatacttgttagaattttactgatttttcacatatatatctatgacccatgtcgaaaatactatagctcatgtgcta aatacattagtaccattgttttgtaattgttttggttttggaacaggattgaaactgaagggaaaggggtaatgaaaccagttgatctcttgaaccatttggttctgttactagtaaaacaaatggagtaaatgttgtacctagtgcacttgtggaagttctcagttgcagccaagaagctgtgtgattgtaccaccaaaactagcactagctgtacgt ccgaggcccggtgtatgggagtacttgtcactgaatcttaagacaaagaaagtggctgaattgagcattcctgagtaccttcaattgaaagagaatactgttgatgaaaggtaaagtaatagtctgcgatttcgctttgtgaaattgaagtttttgtttgattcttaatgttttgtgtatcaattatgttaccagtggaaacatctct tggagttggattttgagccatttacaactgttacaacaccaaaaacactttctgactctattggcaatggtttggagtttcttaatcgccacattgcttcgaaaatgtttcttgataaggagattgccaagtgcctccttgactttctcagaaaccataactacaaaggaaaggtagtaaaaaaagtgtttctttaaacaagttgtatg attatgtgtgtatttctaaatatgtcaatttgaaaacagtcattgatggtgaaagaaagcattcaaagcctggagagtttccaacttgttctgaaaaagcagaggaatatttgcacacactgaatccagaaactccatactccaaatttgaatccaagtttgaagagattggcttggaaagagggtggggaaacaccgctgaacgcgtgca agacaccattagtcatcttttgcatctccttgaggctcctaacgcgtcttccttggaaaatttccttggtagaatcccattggttttcaatgttgtgattctcaccccacatggttattttgctcaagataatgtcttgggctatcctgacactggtggccaggtttgtgtccgataacatatcaagaaattttgcattcttgatcatgttctttataccat ttgaaccaacattctttttttggttgtgaaatgttgaataggttgtttacattcttgatcaagttccagctatggagcgtgagatgcttcatcgtatgaagcttcaaggactcgacgatatcatccctcgcatccttgttgtaagtgcccttaattttcctggtttggtttacctctaaatgaaattgattttctggctttctaacttttttgg attgatctttttgttgttttatcaggtaactaggctgctgcctgatgctgtaggaaccacttgtggcgagtggatggagaaagtatggggcagaacattctcatataattcgtgttccatttagaactgagaaaggaatgttgcgcaaatggatctcacgattcgaagtctggccatacatggaaactttcactgaggttggaacataaaaac aaataaaaatcattggaatgttcttctgcatttgaaaatgtcttgctaactaaagactcatttttaaattaatcatcaggatgttgcagaagaacttgtcaaagaattgcaagctaaaccagacttgataattggaaactacagtgagggaaatcttgctgcctcattgcttgctaagaaatttggggctactcagtgtactattgctcat gccttggaaaaaactaagtatccaaactctgaccttaattggaagaagtttgatgacaagtatcatttctcaagtcagttcactgctgatctttttgccatgaatcacactgatttcattatcaccagcactttccaagaaattgctggaaggtaaaagcaaatgcacaccatcatagtatttcatatttttaccctagtttatactatttccatttgtcaact ccaacttgtttgggattgaaccatagttgttgtttgtttatactatttccattcgccgaccccaacttatttgggactgagacataattgttgttattattgtttgtttgtttatactatttccattctcagaccccaacttctttgggactgagccgtagattgttgttgttgttgttgttgttgttgttgttgctatttccgttcaccgaccccaacttatt tgggactgaggtgtagaagtagtcgttgtagaagtagtcgttgttgtttgtttatacgacttccaattgatattcgaatgtttttatttttgcagcaagaacactgtaggacagtatgagagtcatactgcttttaccatgcctggattgtatcgagtagtccatggaatcaattcgtttgatccaaagttcaacattgtctcccctggggctgatatgtcaat ctacttcccttacactgagaaggagaaaagactaaccaacttccacccggaaattgaagaactcctctacagtcctgttgagaataaggaccacttgttagtctctctttttcattcatttttctacacctttttttcaacagattgattgattggttcttatcaacgtaaacagatgtgtgttgaagccagaacaagccaattctctttaccatggcaaggctag atcgcgtgaagaatctaacagggctcgtggaatggtatgcaaagaatgcaaggctaagggagctcgttaaccttgtggttgtaggcggagacagaaggaaagaatccaaagatttagaagagcaagcagagatgaagaagaagaatgtatgatcttatcgaaacatacaacctgaatggccaattcaggtggatttcttcccaaatgaatcgtgtga ggaacggagaactttatcgatacattgcagacacgaggggtgctttcgttcaaccagcattttatgaggcatttggtttgacagttgttgagtctatgacttgtggtttgccaacttttgctacttgtaatggtggaccatttgagattatagtgaatggaaaatctggtttccatattgatcctaatcaaggtgacaaggctgctgatatgttggtta atttcttcgaaaaatctaaagaagatccaagttattgggatactatttccaagggtggtgcagcgtattcttgaaaagtaagcttttgcatttgattagcacaagtgtacaaccaagatttaacttatgaacaaactaaaactaaccctttttttattttcttttgctaggtacatggcaaatttattcacagaaagtgatcacattatctggga tttatggattctggaaatatgcaaccaagaatgacaaagttgctagtgcgaagaagcgctatcttgaaatgttttatgaatttgggtttaagaaatcagtaagtgtcacttctgtattttgtttgagcttgttgtaaagtttggcaatcttctgctaatttgtactatatttgttgacttgtgcatttcaggctgagaaagttccatt ggctattgatgaatag

SEQ ID NO: 4: NtSUS1-T polypeptide sequence

MAGSGLSIKESLEESILAHPDEILALKSRIETEGKGVMKPVDLLNHLVSVTSKTNGVNVVPSALVEVLSCSQEAVIVPPKLALAVRPRPGVWEYLSLNLKTKKVAELSIPEYLQLKENTVDESGNILELDFEPFTTVTTPKTLSDSIGNGLEFLNRHIASKMFLDKEIAKCLLDFLRNHNYKGKSLMVKESIQSLESFQLVLKKAE EYLHTLNPETPYSKFESKFEEIGLERGWGNTAERVQDTISHLLHLLEAPNASSLENFLGRIPLVFNVVILTPHGYFAQDNVLGYPDTGGQVVYILDQVPAMEREMLHRMKLQGLDDIIPRILVVTRLLPDAVGTTCGEWMEKVYGAEHSHIIRVPFRTEKGMLRKWISRFEVWPYMETFTEDVAEELVKELQAKPDLIIGNYSEGNLAASL LAKKFGATQCTIAHALEKTKYPNSDLNWKKFDDKYHFSSQFTADLFAMNHTDFIITSTFQEIAGSKNTVGQYESHTAFTMPGLYRVVHGINSFDPKFNIVSPGADMSIYFPYTEKEKRLTNFHPEIEELLYSPVENKDHLCVLKDQNKPILFTMARLDRVKNLTGLVEWYAKNARLRELVNLVVVGGDRRKESKDLEEQ AEMKKMYDLIETYNLNGQFRWISSQMNRVRNGELYRYIADTRGAFVQPAFYEAFGLTVVESMTCGLPTFATCNGGPFEIIVNGKSGFHIDPNQGDKAADMLVNFFEKSKEDPSYWDTISKGGLQRILEKYTWQIYSQKVITLSGIYGFWGFKYATKNDKVASAKKRYLEMFYEFKKSAEKVPLAIDE

SEQ ID NO: 5: Polynucleotide sequence of NtSUS2-2

atggctgaacgtgctctgactcgtgttcacagccttcgtgaacgtcttgatgccactttggctgcacatcgcaatgagatattgctgtttctttcaaggtattgcctaagtagtgttcttgtttcctacaaaagattcagttggtgttcaaaaaacgatatgtgatttgatttatctgcctaagtcttggtagtcataattat ccggtacctgtgctggtgcgagttagctggttcggaaactactcttatgaaaacgagagatttagttggtgttgtctgcaattctgtagtatggactattaagcagatagatcatgtttgatatcgaaaaggaatgtatatgtgatgttacttgaactggttttggttattacaggattgaaagccatggaaaagggatcttgaaacctcaccagct attggctgagttcgatgcaattcgccaagatgacaaaaagaagctgaatgatcatgcatttgaagaactcctgaaatctactcaggtaattttgattttggctaaatgtgttaccaagctgaatgatcatgcatttgagtttgtgtccgactactactacaatgatgttataccaggaagcgattgttctgccaccttgggttgcacttgccattcgt ttgaggcctggtgtgtgggaatatgtccgtgtgaatgttaatgctctagtcgttgaggagctgaccgtccctgagtatttgcattttaaggaagaacttgttgatggaacgtaagttttagtctcttatttgatactatgttagagaataggcagtggattcaatttatcagtgttgtttttacctaatgcagctccaatggaaattt cgttctcgagttggattttgagcccttcactgcatcctttcctaaaccgaccctcaccaaatctattgggaatggagttgaattcctcaataggcacctttctgcgaaaatgttccatgacaaggaaagcatgaccccgcttcttgaatttcttcgggttcacaattataagggcaaggtaactttgtttattcccattcatatatatgttcagtttgt gcttatcatgcgcccaatgatgtatgaatatgtactaaaggatagatgtacgatttcgtttgcagacaatgatgctgaatgacagaatacagaatttaaccactctgcaaaatgtcctaaggaaggcagaggaataccttattatgcttccccctgaaactccattttccgaattcgaacacaagttccaagaaattggattggagaagggatggggc gacactgcggagcgcgtgctagagatgatatgcatgcttcttgatctacttgaggctcccgactcctgtactcttgagaagttcctagggagaattcctatggtgttcaacgtggttatcctttccccccatggatatttcgcccaggaaaatgtcttgggttatcccgacactggtggccaggtgcattactttagtctttgtccgt gagtctatgttgctcagatcctctacaatgccactgtacccgtgtaggatactccaaatataatgcatttttggaggatctgtcaccggtgcaatggcattttggaggtcggagcaacaaacaactgctagtatgcttctaaagcttgcttccataaatgctaaggtccttcacccgtaatgtgcaggttgtctacatattagatca agttccagccttggagcgtgaaatgcttaaacgcctaaaggagcaaggacttgatataacaccgcgtattcttattgttagtattctcttgtacttgtaattgctgcggattacacaaaattttctctttattggcaacttatcttgatattattcccaggttactcgtctgctgcctgatgcagttggaacaacttgtggtcagc ggcttgagaaggtgtatggagccgagcactcacatattcttagggtcccctttaggaccgagaagggcattgttcgcaaatggatatctcgctttgaagtgtggccatacatggagactttcactgaggtgacactaagcttccttgtatttgtcttcttctaattggtattaggaacaatttgctaattattaacgctttggcttttcgtacatcagg atgttgcaaaagaacttgctgcagaactgcaggccaagccagatttgataattggcaactatagcgagggaaatcttgtggcttcattgctggctcacaagttaggcgtaacgcaggtctgtgttatttttcacctcttataaatctgattgtatttccattagtctggaactaaaagtactaaaattttcttttcttcgctgtgttat ttgccttctgcagtgcaccattgcccatgcattggagaaaacaaagtatcctgattctgacatctactggaaaaaatttgacgaaaaataccatttctcgtcccagtttaccgctgatcttattgcaatgaatcacaccgattttatcatcaccagcactttccaggagatagcaggaaggtataacatcaattgctaattcggttgcagtaacattt tgttcgatttcttccccttatgcttaacctaataccctaatgaattttccagcaaggacactgtcggacagtacgagagtcaccaggcattcacaatgcctggattgtacagagtcgttcacggcattgatgtgttcgatcccaaattcaacattgtctcacctggagctgatataaacctgtatttcccatattccgagaaggaaa agagattgacagcacttcacccagaaattgaggagcttctgtacagtgatgttgagaacgaggaacatctgtaagtttctaacttactcgtaccgtcagtggcagagccagaattttcattaaaatggggtcaaaatataaagacataaattcacaaagaagccaaggggtgtcaatatgtagtaaaatatattaaaaaaattacctagctacaca atgtaattttccgacaaaaggggtatcggttgcacttcttgaatacatgtggctctgccactgggtacagttacaaagtcctgttacctatgtagatgagcttgtgctgaacatgttgtgattttggtaggtgtgtgctaaaaggacagggaataagccaatcttattcacaatggcgagattggatcgtgtgaagaacttaaccggacttg ttgagtggtacgccaagaacgcacggctaagggagttggttaaccttgttgtcgttggtggagaccgaaggaaggaatccaaagatttggaagagcaagcagagatgaagaagatgtatgagctaataaagactcacaacttaaatggccaattcagatggatttcttcacagatgaaccgagtaaggaacggcgaactctaccgatacattgccgacactag gggagctttcgtgcagcctgcattctatgaggctttcggtttgactgttgttgaggccatgacctgtggtttgcctacatttgcaactaatcatggcggtccagctgagatcatcgttaacggaaaatccggcttccatatcgatccatatcacggtgagcaagctgctgatctgctagctgatttctttgagaaatgtaagacggaac cttctcattgggaaactatttcaaccggtggcctgaagcgcatccaagagaagtaagcaactctttcttgactctagtcattcaaattaacttgggatttgaggcatagttgattgataatttatcgcgtctctactactatacaggtacacgtggcaaatctactcggagagattattgacgttggctgctgtttacggtttctggaaacatgtttctaagctt gatcgtctagaaatccgtcgatatctagaaatgttttatgctctcaaataccggaagatggtgagttcttctgcttcctgctcttctcatagtgtttaatatacacttgattgattgcattcacttagactaagttgctcggacaccgggtgtggatgtccgaccgagtgcggatctagagttcagatccttcaagatgtaaattataagatt cggggatatggatcctagtacggatacgggtgcgagaatccggctaaaaataattttaaaaaaaattatctctaaattatgagatattatgtggaatacttacgtataacttgtaaagtgtagattttttttaattctcaagttgtagattagattagtaaatgattgatttcctagataagtatgctattttcttcaaatttactcttctgatttcgaaa atcaaattgtatctcgtctcgaatttttccgtccgttatggtcaaagtacccaaaatcgtttgaccaaatcggtacggatcccatacccacacccacactagtgtcgtattgacaccggtgccgcacctaaactgctatgtcggagcaacttagcacttagagaatcattgatgttaaattttcttaattcttgaatctgctaatgaaga ttttatcttggttttgtttaggctgaagctgttccattggctgctgaatga

SEQ ID NO: 6: NtSUS2-2 polypeptide sequence

MAERALTRVHSLRERLDATLAAHRNEILLFLSRIESHGKGILKPHQLLAEFDAIRQDDKKKLNDHAFEELLKSTQEAIVLPPWVALAIRLRPGVWEYVRVNVNALVVEELTVPEYLHFKEELVDGTSNGNFVLELDFEPFTASFPKPTLTKsignGVEFLNRHLSAKMFHDKESMTPLLEFLRVHNYKGKTMMLNDRIQNLTTLQNV LRKAEEYLIMLPPETPFSEFEHKFQEIGLEKGWGDTAERVLEMICMLLDLLEAPDSCTLEKFLGRIPMVFNVVILSPHGYFAQENVLGYPDTGGQVVYILDQVPALEREMLKRLKEQGLDITPRILIVTRLLPDAVGTTCGQRLEKVYGAEHSHILRVPFRTEKGIVRKWISRFEVWPYMETFTEDVAKELAAELQAKPDLIIGNYSE GNLVASLLAHKLGVTQCTIAHALEKTKYPDSDIYWKKFDEKYHFSSQFTADLIAMNHTDFIITSTFQEIAGSKDTVGQYESHQAFTMPGLYRVVHGIDVFDPKFNIVSPGADINLYFPYSEKEKRLTALHPEIEELLYSDVENEEHLCVLKDRNKPILFTMARLDRVKNLTGLVEWYAKNARLRELVNLVVVGGDRRKESKDL EEQAEMKKMYELIKTHNLNGQFRWISSQMNRVRNGELYRYIADTRGAFVQPAFYEAFGLTVVEAMTCGLPTFATNHGGPAEIIVNGKSGFHIDPYHGEQAADLLADFFEKCKTEPSHWETISTGGLKRIQEKYTWQIYSERLLTLAAVYGFWKHVSKLDRLEIRRYLEMFYALKYRKMAEAVPLAAE

SEQ ID NO: 7: NtSUS2-T polynucleotide sequence

atgctttttatgggagtaaattttatggccggtcattcaactttgtgttcattacgcaaaagtcatttttcttggtgtttattacgcaagtcatttttcttttttttgttacgtaaaatcattcaactatgtgtttattatctaaaattcaatttttttttcctttgttacacaaaatcattttactttactctttttatcacaaa agtcaccttggccagattttataataggcttttatcttttgttacacaaaaattattttactttactctatttatcacaaaagtcaccttggccagattttataataggcttttcttttgttacacaaaaattattttactttactctatttatcacaaaagtcaccttggccagattttacaatacttttaccttaaaagactattatgcccttgacattataaatcctctcat ttatataataccttctattgatacactatataatattttttacctaggtattttacttataattaaaataatattaaattatttttttttctattttataatatattcatacatttaattttttcatggcaaatcactttgtttaatcatatttaaacatgaacaaattttaaatatatatcaaaaaaataaaaaaataaaaaaataattttttgaaataataacaaacagattt gtttaacaaatgatagtttttttttatagtcaataaaatttttaaaaaaattcaaagatatttgtttttaatattaatatttttaaagcttttatctgttaatattatttatttgaaagtattaatctgatgtgtcattgtgttaaatgtgagtattttatttattggattaatgagtatggcttggctgataaaaagctttgattttataattttcat taaaaatattttattaagctagtacctgacaaatttaatatcttgaaaattaacgttaagaaaaaattaaatataaaaatatattataaaaataataaataaataatatcaagttattttaattataaataaaatacatggttaaaaatatattatatagcatataatatagaaggtattacataaatgagatgatttaaagggcataatagacttttcaggtgaatgatttgta aaatatggttaaagtgattattgtgataattagagcatagtaaaataatttttatgtaacaaaagaaaaaaaaaatgacttttgggtaatgaacataaatttgaataactttacgtaacaaaagaataaaataaattttggataataaacataaaattgaatgaccacctataaaatttattattttttgggctcttcttgatttgattttttagtt tagcctttgcagtaatcttggttgtcacgcgtagcgttgtgctttcgccacataagtatttagtagacttaattaatgtcattatatcggttggtgtggttttaattacttaactgtactattatattaggtggaaggtttgaaaatttatagtagtaacattctagatcattgaaaattggtgtttcagtgactttttagtatgtcattttcatttt ctaagtggttgtactaatatagtatattaaaattttgattggttgagaaacaatctctctcacctacacggtacgggtaaggtatgcgtatacgcttatcctccctacactccatttgtgggactattgttgttattttggataagctgaggtatccatcttctactaactgcactagtttattttttttgctgtttacagttgaaacaattgt ctgaggatttctcacctgctgaatcaactgcaatggctgaacgtgtgctgactcgtgttcacagccttcgtgaacgtcttgatgctactttggctgctcatcgcaatgagatattactgtttctttcaaggtatagccaaagatagtattcttgttaactaaaaagattcagttggtgttcaaaaaacgatacgtttatctgcctaag tcttggtagtcagaattatccggtacctatgctggtgtgagttagctggctaggaaaccactcttatgaaaacaagagagatttagttagagttgtctgtaattctgtagtatggactatgtatgtgatgctatttgaactggttttggttattataggattgaaagccatggaaaagggatcttgaaaccgcatcagctattggctgagtttgatgca attcgccaagatgacaaaaagaaactgaatgatcatgcatttgaagaactcctgaagtccactcaggtaatatggttttggctatatttgtcgccaacgccaagctcatatttttatattattttgagcttgtgtctgaatacgacgatgatgatgttatactaggaagcaattgttctgccaccttgggttgcacttgcgattcgtttgaggcctggtgt gtgggaatatgtccgtgtgaatgtcaatgcgctagtcgttgaggagctgactgtccctgagtatttgcatttcaaggaagaacttgtcgatggaacgtaagtgttagtcttcaatttgatgctatgttagagaataggctgtggaatttattgatcaatgctgtgctttgtcctgatacagctccaatggaaatttcgtt ctcgagttggattttgagcccttcaccgcatcctttcctaaaccaaccctcaccaaatctatcggaaatggagttgaattcctcaataggcacctctctgcgaaaatgttccatgacaaggaaagcatgaccccgcttcttgaatttcttcgggttcacaattataagggcaaggtgacttgctatttccatttatctataggttcggtttgtgct tatcatgcgcccaatgacatatgaatatgcgctaaaggatagatatatgatttcctttgcagacaatgatgctgaacgacagaatacagaatttaaccacactgcaaaatgtcctaaggaaggcagaggaatacctcattatgcttccccctgaaactccattttccgaattcgaacacaagttccaagaaattggattggagaagggatggggcgacactg cagagcgcgtgctggagatgatatgcatgcttcttgatctcctcgaggctcccgattcctgtactcttgagaagttcttggggagaattcctatggtgttcaatgtggttatcctttccccccggatatttcgcccaggaaaatgtcttgggttatcccgacactggtggccaggtgcattactttaatctttatccgtgagt ctatgtttgttcgaatcctctagaaatgtcactgtacctatgtaggatactccaaatataatgcattttggggggatctgttatggggtgcgatggcatttttggaggtcggagcaacaaacaattgctatgtattcttctaaagcttgctttcataaatgctaaggtccttcacccttaatgtgcaggttgtctatatattagatcaag ttccagccttggagcgtgaaatgcttaagcgcctaaaggagcaaggacttgatatcacaccgcgtattcttattgttagtatttcctgtacttgtaattactgcggattacacaaaatttcctttttatcttcttaacaacttatcttgatggtattcccaggttactcgtctgctacctgatgcagttggaacgacttgtggtcagc ggcttgagaaggtgtatggagccgagcactcacatattctgagggtcccctttaggactgagaagggcattgttcgtaaatggatctctcgctttgaagtgtggccatatatggagactttcactgaggtgacactaaaacttccttatatttgtctatcttctaattggtattaggaataatttgttaattgttaactctttgtcttttcgta catcaggatgtcgcaaaagaacttgctgcagaattgcaggccaagccagatttgataataggcaactatagcgagggaaatcttgtggcttcattgctcgctcataagttaggcgtaacacaggtctgtgttgtttttcactctcttaaagatctgattgcatttccattagtctggaactagaagtactaaaaagttcttttcttcactgtg ttatttgccgtcggcagtgcaccatagctcatgcattggagaaaacaaagtatcctgattctgacatctactggaaaaaattcgatgaaaaataccatttctcgtcccagtttaccgctgatcttattgcaatgaatcacccgattttatcatcaccagcactttccaggagatagcaggaaggtataacatcaatttgctacttcgactgcaacagcat tgtgttcccatttctttcccttatgcttaacctaataataccgtcatgaattttccagcaaggacactgtcggacagtacgagagtcatcaggcattcacaatgcccggattgtacagagttgttcacggcattgatgtgttcgaccccaaattcaacattgtctcacctggagctgacataaacctatttcccatattcgagaaggaaaagag actgacagcacttcaccctgaaatcgaggagctgctgtacagtgacattgagaacgaggaacatctgtaagtttctaccttactcgtacagtcagtggcggagccagaattttcactaaaataaggtcaaaatataaagacataaatccacaaagaagccaagggtgtcaatatatagtataaatacattaaaaaaattacctatctacacagtgtaat tttccgacaaaggggtgtcggttgacactccttgaatacatgtggctctgccactgggtacagttacaaagttctgttacctatgtagatgagcttgtgctgaacatgttgtgattttggcaggtgtgtgctaaaggacaggaataagccaatcttattcacaatggcgagattggatcgtgtgaagaatttaaccggacttgttgagt ggtatgccaagaacgcacggctaagggagttggttaaccttgttgtggttggtggagatcgaaggaaagaatccaaagatttggaagagcaaacagaaatgaaaaagatgtatgagctaataaagactcacaatttaaatggccaattcagatggatttcttcacagatgaaccgagtgaggaacggtgaactctaccgatacattgctgacactaga ggagctttcgtgcagcctgcattctacgaggctttcggtttgactgttgttgaggccatgacctgtggtttgcctacatttgcaactaatcatggcggtccagctgagatcatcgttaacggaaaatctggcttccacatcgatccatatcacggtgagcaagctgctgatctgctagctgatttctttgagaaatgtaagacagaaccttct cattgggaaaccatttcaacgggtggcctgaagcgcatccaagagaagtaagcaactctttcttgactctagtcattgaaattaactttcttgactctagtcattgaaattaactcgggatttgaggcgtagttgattgatatttttcgcgtctctactactactgatatacaggtacacgtggcaaatctactcggagaggctattgacattggctgctgtttacggggtt ctggaaacatgtttctaagcttgatcgtctagaaatccgtcgatatcttgaaatgttttatgctctcaaataccgcaagatggtgagttcctcttcttccttgcccttctcctagtgtttaagatacaatataattgattgcattatcttagagaatcattaatgttaaattttcttaattcttgaatctgttaatgaagttttctcttgg ttttgtttaggctgaagctgttccattggctgctgagtga

SEQ ID NO: 8: NtSUS2-T polypeptide sequence

MLFMGLKQLSEDFSPAESTAMAERVLTRVHSLRERLDATLAAHRNEILLFLSRIESHGKGILKPHQLLAEFDAIRQDDKKKLNDHAFEELLKSTQEAIVLPPWVALAIRLRPGVWEYVRVNVNALVVEELTVPEYLHFKEELVDGTSNGNFVLELDFEPFTASFPKPTLTKsignGVEFLNRHLSAKMFHDKESMTPLLEFLRVHNYKGK TMMLNDRIQNLTTLQNVLRKAEEYLIMLPPETPFSEFEHKFQEIGLEKGWGDTAERVLEMICMLLDLLEAPDSCTLEKFLGRIPMVFNVVILSPHGYFAQENVLGYPDTGGQVVYILDQVPALEREMLKRLKEQGLDITPRILIVTRLLPDAVGTTCGQRLEKVYGAEHSHILRVPFRTEKGIVRKWISRFEVWPYMETFTEDVA KELAAELQAKPDLIIGNYSEGNLVASSLLAHKLGVTQCTIAHALEKTKYPDSDIYWKKFDEKYHFSSQFTADLIAMNHTDFIITSTFQEIAGSKDTVGQYESHQAFTMPGLYRVVHGIDVFDPKFNIVSPGADINLYFPYSEKEKRLTALHPEIEELLYSDIENEEHLCVLKDRNKPILFTMARLDRVKNLTGLVEWYAKNARLRE LVNLVVVGGDRRKESKDLEEQTEMKKMYELIKTHNLNGQFRWISSQMNRVRNGELYRYIADTRGAFVQPAFYEAFGLTVVEAMTCGLPTFATNHGGPAEIIVNGKSGFHIDPYHGEQAADLLADFFEKCKTEPSHWETISTGGLKRIQEKYTWQIYSERLLTLAAVYGFWKHVSKLDRLEIRRYLEMFYALKYRKMAEAVPLA AE

SEQ ID NO: 9: Polynucleotide sequence of NtSUS3-S

atggcgaatccaaagttcacaagagtacctagcatgagggagagttgaggatactctctctgctcaccgtaaccagcttgttgctctcctctccaggtatattaataaactctatatacttgttattttctttattttttgtctttactgataaatttaactgttttcttctttaaatcttgctttcgatgcatgatttctgttgtgttaaattgc gtaaccattttttctaaaagttttgctgataaacacttttaaattttaatatgtaaattatattatgtctcaacatcaacatgtggatggccaaaaatataaagcttaattttcgttattttgaatgatttttctctgcgagtgttacggtttgcgtacacattacctaaacctcctccctagtccccacttgtgggaatttaatttttttt ttctttgtttttttttgttgttgttgttgtctgagttcaattcctaccatgttagcttggcaaaaataagttggtaaagcttgaccccaactagttttagttgtcgatttatttggtgatttatagttcaataataattaattactattagagaaagttccagcagctttctgtttgtttttccagttttagtgattgatatatgtgtatatatattctttg tttcttttaagatacgtggcgcaggggaaggggatattgcaacctcaccacttgatcgatgagttcaacaacgctgtatgtgatgacactgcttgtgagaagctcaaagatggtccctttagtgaagtcttgaaagctactcaggtatattcactaatccatgggaatcaagatgatactgtatctttttattatggtgtctttcagaaattt gacgatgatgaaatgcaacttttctctgtttgtcaccttatccagactgtttttttattttttatttttcattttttaacttgaaatgctcttaatttcctttgttttcgataagaccggatttacaatgtatgaacggagcatcttaagaaccttctctggaatgaagatataagatataaaacatggtgtccgttttctcctttgtggaatcagtgta catatagactgtttattttggtcccactttctggatcttcttgatcacaccttctcatgcagaggcgagcttgatggtttcaacctttaaattcttactattgaatccatttcactttcgaaattatgagttcgaaatctaatatttgttgaaatttttgcaaatgttcacatataagtttaagctttgtgtcaagaatactgggctcaatggattccaatagacca ggctgtatccgcctctgtctccactctccctgcatccacttctttcgtgtgactaataatgcttaatgagctagaactcgttttaatgtttgaataagttgcttatatcagagcagcttttgatgtttcaatctttaacgggttatgcagtaccagcattctgcggctgaaaaacaggaatctgagatttacttgtctctggctgaatttct tgttcattttgctaacaagtactttggagttaatgcttgctctctgttgtcaaaataggaagccattgtgctgccaccatttgttgccatagcagttcgtccaaggccaggtgtttgggagtatgttcgtgttaatgtatgatttgagcgttgaacaattgactgttcctgaatatcttcatttcaaggaagaacttgtggat ggagagtaagctctttcttatttcaatacgaaacataaaaatttacagaagttgaataattaattaacaaatttgttgatttttaatgtatgccaggggtaataatcactttgtgcttgagctggattttgagccatttaatgcatcagttcctcgtccatctcgatcgtcatccattggcaatggagtccaattcctcaatcgtcatctttcctcaattatgttt cgcagcaaagactctctggaccccttacttgatttccttagaggacactgtcataaagggaatgtaagtaccaaaagcagttttccctttgtaaatgtctgcttgtccctgattatctactaaatctttcaacacgcgcaaccattataagaaatgtacaatacttctagttagaatttcatcgacaaactatctgctttactttttatttttcccatttgat ggatgatagtttagtttatataacagatgatattttggttgaagggtaccatgaactttttcacaaccacttaatggatacatagttgtaatagttgacattttggaataatattgtctcacttggaaatgtttaagaagtattactacttctatttgtaagatggattgtttatctatgcaggtcttgatgttgaatgaatgatcgtatacagcgaatctccaggctggagtctg ctctttctaaagcagaggattatctctccaagctatcaccagatacatcctataatgagttcgaatacgcgtgagcttgtacacatttgttttgttttctttcaagcatatgtaatttctcaagaaaagggaaatcttaggagttgaaacattctttttggaaccatgtgcatgcagattgcaagaaatgggctttgagagaggttggggtgatactgccagacg tgttttggagacgatgcatcttctttctgacattcttcaggctccggatccatcaaccttggagacatttcttggtagactacctatggtgttcaatgtcgtcatattatcccctcatggatattttggccaagcaaatgtcttgggtttgcccgacactggtggccaggtaataacaaggagaatgaggtcttgtattatgtactccctccgttccaatctatga acctatttgactgggtatggaaagaaatgaagacttgtaaaacttgtggttctttagaaattccaaacattacatttggttttttcccttcctggaaattatactactgaatcatctctagatgttccagtttaacttgagacgtaagggtaaataacggaccattactctgtcctttcttgcagtaggcttggtacaatgaatatagttcgcatagttgccggaag ctagagctgtgttagaaaactcaggaacattaatttggcgatgctaatcactgctaatgttactgaagcatccatggttttccttgatgttattctccttttggttgcttcacaggttgtctatatactggatcaagtgcgtgccttggaggccgaaatgcttcttagaataaagcaacaaggacttaacttcaagcctagaatccttgtcgtgag tacatatatattatgcaagctcttatttggtttgtgggattgcagttgacatcaatttgcttactctgattactaaaggtcacacggctgatacctgatgctaaaggaaccatgtgcaaccagaggttggagaggattagtggaactgaatactcgcatattttacgtgtcccttttaggacagagaagggaatccttcataaatggatatctaggtttgat gtatggccttacctggagaagttcactgaggtaacctctttgtccctttggaaattgccttttgttgctgatgtttctgctagtgtgcttaaatgacggatgttaactagtcacttgctagcgtttgcaatagcaacgggaaaagaaaggattttgctagtttgaagtctgcctccaagaaaaattatattaaaagtttatggctagt ggaaacatcagtcattcatgtaccttatttctatgcccaagttgtttaagttgaaagtaatttggccaactatgcaaattgggagaacgtgtagccaactattgtgtttgccgacatgttgatatactttttggtcctgatttatatttgttggtttgtcatactggatgaagcaattctcatgttttctgcttatatatattggaagaagaagatacttgt cgtttcatcatttttctcgacctctctattaccaacactttgccaatttaatgtttggaaatgtcttcttgaccaggatgtggcaagtgaaatgaccgctgagctccagggaaagccagatctgattattggcaactacagtgatggaaatttagttgcctcccttttggcatataaaatgggtgtcacacaggtaggaaatacatgattcttttcttgctag cactaagtcttgaggttatgtatctgcaatagaaattttacgctttgccttcatttcttttaattattttccagtgtaccattgctcatgccttggaaaaaacaaagtatcctgattctgacatctactggaaaagtttgaggagaaatcatttttcatgtcagtttactgctgatctactggcaatgaataattcagatttcattatcaccagt acttatcaagagattgcaggaacgtaagtcattttaatctggtcgtttaaatctgatatttcttccctagtagtctattcaatccgaatttcagttcagtatatgatgtcatcggttgaggaactgtgattggtaaccttatcaaatccgtagctgctctataattttattcgtaatttggagaaacaatttttattattgagcttgtagtctgagct agaatttggttctttatctatcaagtagcataatactacaactattttttatgtgtggcaatttgcaatttcaattttctatttctataagttgcagcttttcttcctgttctgatcatatttacatggctgaaactcaatagaaaactaggctagttgatcaaaagtagttggatgctttaaaattagtagacgttttgctaaatgagtgaccaatgttattaaa aaaacgttcatgttttcaacccttttggcatacatttgaccactgcccaagattttggataagtacatgcagtgcttataattataaagcatttttcccaccttgtttttcattatgaaaattaagtaatttacgagtatttgtataagttacttcataaattagaagtaaatctggattgtgtaaagttattcgccccgtatatactgaaagctacttgaacaag caaaaaaacagacaaacgtaacattctccatggattaatgagacttgtatatatatatatatatatatgtaaagagagagagagagagatttggcttgtaaccacatgtatattatgccatatggatgtgacattgatgtgactagacctaaatgttttgtttcaatgtccacgggagttttacgtagagttaagaggagaagagagtgaggaatactaat gtttgatggtaccccttggcttcttgacctggatactcagtgttcttattcatgcctatactttggtccttgatttcattctcccttttctagcttgagctgcatcaaagaaattccactgtaaaaaaaataatgctcaccatattggtgcaacatggcaaacatgtatcctattgatgatcaatcaactttatttttctcctgttaattgacc tcagtgtgtaactctctatgtatgatagcattgtaacttgtgtcatgattcataaatagggtactagaattggatggttgacatagtaaatggtcaattgatgatccacaaaatatgcacctactgattaaaatgtgatagggcaggtttatttttgtttgtggttaacacagtacttaaccctatatttaatacaatttggcttatctacaatcttttt cttcagtgtttatgcgaattccttattgcacaacaatattgtctttctgagttctattctgttgttgcttacacttttttccagtaacatagatgtgaagacattagattggttgcttgcaaattgatagccacttgtttcaggaagaatactgttggtcagtacgagccatactgcattcaccctcccgggactatatcgcgtcg ttcatggcattgatgttttcgatcccaaattcaatatagtgtctcctggagctgacatgacaatttatttcccatattctgacaaggaaaaaagactaacgtctttgcatggctcgattgaaaagttgttatttgatcctgcgcagaatgaagagcatccgtttgtacttaatttttttggaatagatgacatattatttgcatgaatat gaaaaggagggtctgatatgattttctatagataaactaccaatgatattatttaaaaactcctggatactgtattaggagaagaagaagaaccaggggtagatggcattagaatcccttaaatcttgaagagtcgtcactaacgctcccaacacttctgcctcagaccctcaactaaatactattattgttgatttctttggagaagctataagaatctctctctccttatggtgaa aattttacttggctttatactacttaacttccaaggctccctcttaaaatgcaaaaactgtctgtattcactctcttggttaacaattgatccaatcaaatgcatatggaacatctttctttacgtttcttctaaaagttcgtttgaggataaggagtagaatctgagaagatagactagtaggtaaccttagggggacggatgtggaaattaacatatgggctcag cttttctgccgagtgcagaccatgtatatgcgttaaaaaattcactaaacaagtaaatgtttgattttgaacccagtaaatcaaatgagttgtggtagaatctcgaactcgaaccgataaagttcaaatccaggatccgcttttaggtaaactctaccttgggaagtgttatatatatgtccctgattatttctttttccgtttcct ttctattttaatttttaaagttatttttagatggttttattttttgataagtggtaagttgttaatattccaaattaaatgccattgtcataactatatacatttataaagaatgattgatcctagtttctcattcctaagatccaaataaggcaataaacaatgtcttagtaattggacctgcttctggtgatcaacgcttgatcgcgtagttagttatagatgactg taaaaactttaaccattttaatggttttgtcaaagaacaaatatcggacatattatagagaatggactattgtactttgcttctgattggtcattttattgtgatccgtaaattggctgtgactgtgtcatatctttgcttacagaggtaatctgaatgataaatcaaaacccataatttttcaatggcaaggctagaccatgttaagaacattacgggactag ttgagtgctatgctaaaaatgccacattgagggaattggcgaaccttgttgtagtagctggatacaacgatgtaaagaaatccagtgatagagaagaaataacagaaattgagaagatgcatgctcttattaaggagcataaattggatgggcaattcagatgggtatcagcccaaacaaaccgggcacgtaatggtgagctatcgctatatag ctgaccagagaggtatatttgttcaggtatgctatttgtattgtattagtccaatttcatttttgcaccaaaagaaaggttgttattgtgacgtatatgtttgttttagcctgcatttttgaagcatttggactaacggtggttgaagctatgacttgtggtcttccaacatttgcaacttgccatggtggtcctaatgagatcattgaacccgg tgtatctgggttccatattgatccttcatcccgataaagctgctgaactcatgtcagaattctttcaacgctgcaaacaagatcctactcactgggaaaaaatatctgcatctggtctccgaaggattcttgagggtctgtagttgtgtacatgtatagaagattaaagaatgctaccttgatatttattgaatcaaaaataacaggaacatctctt ttttgaacatcactcaagttcttatattaaataatttttaggtatacgtggaagatttactccgagaggctgatgactttatctggcgtatatggtttctggaagcttgtttcaaaacttgaggcgtgaaactagacgataccttgagatgttctacattctcaaattccgcgagttggtgagtgcctttagctccttttcagttccaataaact atatatgtggtttaagtaagtattaagcataaacatgtccgtgcttggggctgtcgaaaatgctatggacatatcctgagctaaggatttttcaagaaaattgatgttagctttactctatttacaggcaaaatctgtacctctagcaattgatgacaagtga

SEQ ID NO: 10: NtSUS3-S polypeptide sequence

MANPKFTRVPSMRERVEDTLSAHRNQLVALLSRYVAQGKGILQPHHLIDEFNNAVCDDTACEKLKDGPFSEVLKATQEAIVLPPFVAIAVRPRPGVWEYVRVNVYDLSVEQLTVPEYLHFKEELVDGEGNNHFVLELDFEPFNASVPRPSRSSSIGNGVQFLNRHLSSIMFRSKDSLDPLLDFLRGHCHKGNVLMLNDRIQRISRLESALSK AEDYLSKLSPDTSYNEFEYALQEMGFERGWGDTARRVLETMHLLSDILQAPDPSTLETFLGRLPMVFNVVILSPHGYFGQANVLGLPDTGGQVVYILDQVRALEAEMLLRIKQQGLNFKPRILVVTRLIPDAKGTMCNQRLERISGTEYSHILRVPFRTEKGILHKWISRFDVWPYLEKFTEDVASEMTAELQGKPDLIIG NYSDGNLVASSLLAYKMGVTQCTIAHALEKTKYPDSDIYWKKFEEKYHFSCQFTADLLAMNNSDFIITSTYQEIAGTKNTVGQYESHTAFTLPGLYRVVHGIDVFDPKFNIVSPGADMTIYFPYSDKEKRLTSLHGSIEKLLFDPAQNEEHIGNLNDKSKPIIFSMARLDHVKNITGLVECYAKNATLRELANLVVVAGY NDVKKSSDREEITEIEKMHALIKEHKLDGQFRWVSAQTNRARNGELYRYIADQRGIFVQPAFYEAFGLTVVEAMTCGLPTFATCHGGPNEIIEPGVSGFHIDPYHPDKAAELMSEFFQRCKQDPTHWEKISASGLRRILERYTWKIYSERLMTLSGVYGFWKLVSKLERRETRRYLEMFYILKFRELAKSVPLAIDDK

SEQ ID NO: 11: Polynucleotide sequence of NtSUS3-T

atgtttacatggctgaaactcaatataaaaaacaagggtaggtgatcaaaaatcgttggatgcttaaaatcagtagacgttttgctaaatgagcgaccaatgttattgaaaacgttcatgttttcaacccttttggcatacatttgagcattgcccaagattttggataagtagatgcagtgcttataattttaaagcattgtatcctgcctt gtttttcattgtcaaaattaattaattaacttacaagtatttctataagttgcttcataaattagaagtaaatctggattgtgtaatgtttattcgcctcgtaaatactgaaagctgcttgaacaagtgaaaaaacacagacaaacgtaacattctctccatggattgatgagacttgtaaaatacatatatagaaatttggcttgtaaccacatgtatatta tgccatatggatgtgacattgatgtgactagacctaaatgttttgtttccatgtccactggagttttacgtatagttaagaggagaaaagactgaggaatactaatgtatgatggtacccctttgcttcttgacctggatacccagtgttcctattcatgcctatactttggtccttgatttcactctcccttttctaacttgagctgcatcaaagaa atttccactgtaaaaaaataaataatgctcaccatatctctgcaacattgcaaacatgtatcccatatgattgatattggtgcgacatggcaaacatgtatcctatttgatgatcaatcaaatttatttttcccctgtcaaaatgacctcagtgtgtaattccctatgtatttgatagcattgtaactcgtgtcatgattcatgaataggg tactagaattgcatggttgacaaatattaactggtcgattgatgatccacaaaacatgcacttactgactaaaatgtgatggacagatttatttttgtttgtgattaacacagtacttaaccctatacttaatacaatttggcctagctacaatctttttcttcagtgcaaattccttgttacacgaccaatattgtctttctgagttctattctgttgtt acttacacttttattattcgaataagacattagattgcttgcatgcaaattgatagccacttgtttcaggaagaatactgttggtcagtacgagagccatactgcattcaccctcccaggactatatcgcgtcgttcatggcattgatgttttcgatcccaaattcaatatagtgtctcctggagctgacatgacaatttacttcccatattctgacaaggaaaa aagactaacgtctttgcatggctcgattgagaagttgttatttgatcctgcgcagaatgaagagcatatgtaagtgacatccatttgtacttattttaatttggaatagatgacatacttattgcatgaatataaactgacaacccagagatttcctacattagaaaaggagggtctgatgattttctacaaataaattcccagtgatattgttcaaaa agtcctggatactttattatgagagaaccagggatagatggcactagaatcccttaatcttgagaagtcgccacttatcgctcccaacactttctgagaccctcaagtaactactattattgtttgatatcttggagaagctataagaatctttttctccttattgtaattttttttacgtgactttaaacttaacttccaagctccttctgataaaatgcaaaaactgtctg tattcactgtcttggtttattaacaattgatccaatcaaatgcatatggaacatctttctttttgtttcttcaaaagttcgtttgaggataaggagtagaatctgagaagatagactagtaggtaaccttaggggcggatgtagaaatcaacgtatgggttcagctttgttgcagaccctgtatgcattaaaaaaatcactaaataagtaaataat tgattttgaacccagtaaatcaaaatgagttgtagtagaatcctgaactcgaaccgataaagttggatccactaccgggtaaactctaccttgagaagtgtttatatatgtccctaattatttctttctgtttcctttctattttaattttttaagttcctttttagatggtttttattttttgacaagtggtaagttgttagtattccaaattaaatgccattg ccataactatatacatttataaagattgattgaccctagtttctcattcctaagatccaaataaggcaataaacaatatgtcttagtacttgaacctgcttctggtggtcaacacttgatcgcgtagttagttatagatgactgtaaaaaccttaatcattttaatggttttgtcaaagaacaaatatcggacatattatagcgaatggactattgtacttttcttctga ttggtcattttattgtgatccgtaagttggctgagactgatgtcatatctttgcttacagaggtaatctgaatgataaatcaaaacccataattttttcaatggcaaggctagaccatgttaagaacattacgggactagttgagtgctatgctaaaaatgccacattgagggaattggctaaccttgttgttgtagctggatacaacgatgtaaagaaat ccagtgatagagaagaaaatagcagaaattgagaagatgcatgctctttattaaggagcataaattggatgggcaattcagatggatagcagcccaaacaaaccgggcacgtaatggtgagctctatcgctatatagctgacaagaggtatattgttcaggtacgctgtttgtattgtattgtccacattccttttttgcaccgaaagaaagg ttgtttattgtgacaaatatgtttgttttagcctgcattttatgaagcatttggactcacggtggttgaagctatgacttgtggtcttccaacatttgcaacttgccatggtggtccgaacgagatcattgaacacggtgtatctgggttccatattgatccttatcatcccgataaagctgctgaactcatggcagaattctttcaacgctgcaaaca agatcctactcactgggaaaaatatctgcatctggtctccgaaggattcttgagaggtttgtagttgtgtacatatatagaagattaaagattgttcccttgatattatttgaatgaaaaataacagtaacatctctttttgaacatcgctcaagttcttgtgttaaataattgttaggtatacgtggaaaatttactccgaggctgatgactttg tctggtgtatatggtttctggaagcttgtttcaaaacttgaggcgcgaaactagacgataccttgagatgttctacattctcaaattccgcgagttggtgagtgcctttttgctcattttcagttacaatcaactatatgtggtttaaatacgtattaagcataaacatgtccgtgattgcggctgtcgaaaatgct atggacatatcctgagctaaggagttttcaagagaattgatttggcttactctgtttacaggcaaaatctgttcctctggcaattgatgacaagtga

SEQ ID NO: 12: NtSUS3-T polypeptide sequence

MFTWLKLNIKNKGRKNTVGQYESHTAFTLPGLYRVVHGIDVFDPKFNIVSPGADMTIYFPYSDKEKRLTSLHGSIEKLLFDPAQNEEHIGNLNDKSKPIIFSMARLDHVKNITGLVECYAKNATLRELANLVVVAGYNDVKKSSDREEIAEIEKMHALIKEHKLDGQFRWIAAQTNRARNGELYRYIADKRGIFVQ PAFYEAFGLTVVEAMTCGLPTFATCHGGPNEIIEHGVSGFHIDPYHPDKAAELMAEFFQRCKQDPTHWEKISASGLRRILERYTWKIYSERLMTLSGVYGFWKLVSKLERRETRRYLEMFYILKFRELAKSVPLAIDDK

SEQ ID NO: 13: NtSUS4-S polynucleotide sequence

atggcggaacgtgtgctgactcgtgttcatagccttcgtgaacgtcttgatgctactttggctgctcatcgcaatgagattttgctgtttctttcaaggtatagtcttagcagattgttctttgatttagttgttattgccagttctaatgtatgggcttatatataaacaaagtgttgaagtatgcaaccatataaactgacagctta aaatgcttgagagaacacacttttatttatttaattatgccttcagcacaagaagtggaacttgacgcaatggaaccataggtcacgggttcaagtcttggaacagcctgcaatctaaggctgcgtgtagtagaccctagtggtccggcccttccacatatctcgcttagtgtaccgggcccattgagtacgggttcggccgaacccagtcgct ttggtccaatccatatatttgtcttaaaaatatattgaatatatacaaattgttaatttagtttaaatatgtgtatcatggggttattcatgctggttttggctgttgcaggattgaaagccatggaaaagggatactgaaacctcaccagttgctggctgaatttgattcaattcacaaagaagacaaaaacaaactgaatgatcatgcttttgaagaag tcctgaaatccactcaggtatttgtggttttagtgttaggtgatggatagcatttattgttttactaagatcacatatgtgtcagtttgtggctagtatttaaaatctggtgtattttgtcatactaggaagcaattgttttgtccccttgggttgcgcttgccattcgtctgaggcctggtgtgtgggaatacgttcgtg tgaatgtcaacgctcttgttgttgaggagcttaccgtgcctgagtatttgcaattcaaggaagaacttgttaatggaacgtaagttttaggttcgaatttgttgatttgttagataacatgttctgaactttttgattaaagttgtgtttttgactgatgcagctcgcacgataactttgttcttgagttggattttgagcccttcact gcatcatttccaaaaccaaccctcaccaaatcaattggaaatggagttgaattccttaaccgacacctctgccaaaatgttccatgacaaggaaagcatgacccctcttctcgagtttcttcgagttcaccactacaagggcaaggtaaacttgttttcctgtttgtctatgaatttagtttagttgttttgctccgcgaaaatttcag tggaaactgatttatgcaaccactgagtgattaatatgttcaaacttaccgacttctggttttctgtgtagacaatgatgctgaatgacagaattcaggacttaaatactctccaaaatgtcctaaggaaagctgaggaatacctcactaccctttcccctgaaacttcatactcggcatttgagcacaagttccaagaaattggcttggagaggggttggggtg acactgcggagcgtgttctagagatgatctgcatgctcctggatctcctcgaggctcctgactcgtgcacgcttgagaagttccttggtagaattccaatggtttttaatgtggtcatactttcaccccatggttatttcgcccaggaaaatgtcttgggttaccccgacactggtggccaggtgcactgcttatctgtgttcggtct tattatctctttaaaccctactgccacaagtgctgagatgaacctcctttaatttgcaggttgtctatattttggatcaagttcctgctttggagcgtgagatgctcaagcgcataaaggagcaaggacttgacatcaaaccgcgtattcttattgttcgtattcccagtaattgtgtttaaacttatgattatgcaggattttatctgtt ctaatacagcactcttgcttaaattctcaggttactcggctgctgcctgatgcggttggtaccacttgtggtcagaggcttgagaaagtgtttggaacagagcactcacacattcttagggtcccctttaggaccgagaagggcattgttcgcaaatggatctctcgctttgaagtctggccatacatggagacattcactgaggtgaagcaagctttctct attcatttttcaatcttccaattggttttggcagcaattttctgcttgctttgacttccgctaaaacttcggattttattgcattaggatgtggcgaaagaaattgctgcagaattgcaggctaagccagatcttatcattggcaattatagtgagggcaaccttgctgcctccttgttgctcacaaattaggtgtaacacaggtcggcaatg tttgtgacatgtaatttcatctttgcatttcctttcgtttgcaactaaaagatttaagagttctctctctctttttttttccgtctactttgccttatgcagtgcacgatagctcatgctttggagaaaacaaaatatcctgattctgatatctacttgaagaaatttgatgaaaaataccatttctcagcccagtttactgccgatcttattgca atgaatcacaccgatttcatcatcaccagcactttccaggagatagcgggaaggtatttttacatcagtttcccactctgattaaattacaatgtatttccctatatgattaaatactgtgtttgatcctaaatcatttctaaattttccagcaaggacactgttggacagtacgagagccacatggcgttcacaatgcctggactgtatagagttgttcacggcattgatg tgtttgaccccaaatttaacattgtgtcaccaggagctgatatgaatctctatttttcccatactacgagaaggaaaagagattgacagcatatcaccctgaaattgaggagctgctgtttagtgatgttgagaatgacgaacacatgtatgttactaaactagcaatcctgctgcaaaattatggctaattatgtaaacaagtttgtactgaatagattt gttattcgatcaggtgtgtgctgaagaacaggaataagcctatcatattcactatggctagattggatcgagtgaagaacttaactggacttgtcgagctgtacgccaagaaccccggctaagggagttggttaaccttgtcgtggttgggaggagaccgaaggaaagaatccaaagacttggaagaacaggcagagatgaagaagaagatgtacgaacttataa agactcacaatttgaacggccaattccgatggatttcttcccagatgaaccgcgtgaggaatggcgaactctacaggtacattgccgatactaggggagctttcgtgcagcctgcattttacgaggcttttggtttgactgttgttgaggccatgacctgtggtttgcctacatttgcaactaatcacggtggtccagctgagatcatcgttc acgggaaatctggtttccacattgatccataccacggggatcaggcagctgaacttctcgctgatttctttgagaaatgtaagaaagaaccttcgcactgggaagccatttccgagggcggccttaagcgtatacaggagaagtaagcaaactgctactcttttcatttttgcaaaacctactatgatcattattaagctcatttttgcaaaacctacttgctgttg ttattgtttgttgcttccttttcactgttctttgagctgaaggtctatcagaaacagtctctctaccttcacaaggtaggggtaagatctgcgtgcacgttaccctcctcaaactctacttaattgtgagattacactaggttgttgttgttgattctttgctaattaattaattaaaaggtacacatggcaaatatactcggatcggttgttgac actggctgctgtatggattctggaagcatgtttccaagcttgaatcgtcttgaaattcgccgttatcttgaaatgttctatgctctcaaattccgcaagctggtgagtttcattgctttctgcactcctgcaattgtatag

SEQ ID NO: 14: NtSUS4-S polypeptide sequence

MAERVLTRVHSLRERLDATLAAHRNEILLFLSRIESHGKGILKPHQLLAEFDSIHKEDKNKLNDHAFEEVLKSTQEAIVLSPWVALAIRLRPGVWEYVRVNVNALVVEELTVPEYLQFKEELVNGTSHDNFVLELDFEPFTASFPKPTLTKSIGNGVEFLNRHLSAKMFHDKESMTPLLEFLRVHHYKGKTMMLNDRIQDLNTLQ NVLRKAEEYLTTLSPETSYSAFEHKFQEIGLERGWGDTAERVLEMICMLLDLLEAPDSCTLEKFLGRIPMVFNVVILSPHGYFAQENVLGYPDTGGQVVYILDQVPALEREMLKRIKEQGLDIKPRILIVTRLLPDAVGTTCGQRLEKVFGTEHSHILRVPFRTEKGIVRKWISRFEVWPYMETFTEDVAKEIAAELQAKPDLIIG NYSEGNLAASLLAHKLGVTQCTIAHALEKTKYPDSDIYLKKFDEKYHFSAQFTADLIAMNHTDFIITSTFQEIAGSKDTVGQYESHMAFTMPGLYRVVHGIDVFDPKFNIVSPGADMNLYFPYYEKEKRLTAYHPEIEELLFSDVENDEHMCVLKNRNKPIIFTMARLDRVKNLTGLVELYAKNPRLRELVNLVVVGDR RKESKDLEEQAEMKKMYELIKTHNLNGQFRWISSQMNRVRNGELYRYIADTRGAFVQPAFYEAFGLTVVEAMTCGLPTFATNHGGPAEIIVHGKSGFHIDPYHGDQAAELLADFFEKCKKEPSHWEAISEGGLKRIQEKYTWQIYSDRLLTLAAVYGFWKHVSKLDRLEIRRYLEMFYALKFRKLVSFIAFCTPAIV

SEQ ID NO: 15: NtSUS4-T polynucleotide sequence

atggccgaacgtgtgctaactcgtgttcacagccttcgcgaacgtcttgatgctactttggctgctcatcgcaatgagattttgctgtttctttcaaggtatagtcttagcagattgttctttgatttagttggtgttatttgccagttctaatgtatggactaatatatgaacaaagtgcgaccatttcaactgacaacttaaaatgt ttgagagaatacacgtttatttacttaattatggcttgagcataggaagtgtatcttggcgtaactcgtaaagttgacctcatgtgacaaggaggtcacggtttcgagccgtggaaacagcctcttgcagaaatgcaggtaaggctgcgtgcaatagatcgcccttccacggacccgcgcatagcgggaacttagtgcaccggttgggg ctgtcctttttttgtcttcagcacaaaaatttagtttaaacatgtgtatcatggattattcatgctggttttgccggttgcaggattgaaagccacggaaaagggatattgaaacctcaccagttgctggctgagtttgaatcaattcacaaagaagacaaaaacaaactgaatgatcatgcttttgaagaagtcctgaaatctactactcaggtaattt gtggttttagtgttaggtgatggatagcatttattgtcttactaagatcatatatgtgtcagtttgtggctagtatttgaaaagtctggtgtggtttgtcatactaggaagcaattgtcttgtccccttgggttgcgcttgccattcgtctgcggcctggtgtgtgggaatatgttcgtgtgaatgtcaat gcacttattgtcgaggagctgactgtgcctgaatatttgcaattcaaggaagaacttgttaatggaacgtaagttttaggttcgaaatgatgatttgttaaataatatgttctgaactttttgattaatgttgtgttttcccctgatgcagctcgaacgataactttgtttgagctggattttgagcccttcactgcatcatttcccaaac caaccctcaccaaatcaattggaaatggagttgaattcctcaaccgacacctctctgccaaaatgttccatgacaaggaaagcatgacccctcttctcgagtttcttcgagttcatcactacaagggcaaggtaaacttgtttttcctgtttgtctatgaatttagtttctgaaagttgctttgcttcgtgaattttttagtggcaactga tttatgattttctgtgcagacaatgatgctgaatgacagagttcaggacttaaacactctccaaaatgtcctaaggaaggctgaggaatatctcactaccctttcccctgaaacttcatactcggtatttgagcacaagttccaagaaattggcctagagaggggctggggtgacaatgctgagcgtgttctagagatgatctgcatgctcctggatctcct cgaggctccagactcatgcactcttgagaagttccttggtagaattcctatggtttttaatgtggtcattctttcacctcacggatatttcgcccaggaaaatgtcttgggttaccccgatactggtggccaggtgcactgcttattgtaacaccttacgcttttccctctgaaacttattgcggcaagttctaaggtcctccttcctta atttgcaggttgtctatattttggatcaagttccggccttggagcgtgagatgctcaagcgcataaaggagcaaggacttgatatcaaaccgcgtattcttattgttcgtatctccaataattgcgtttaaacttatgattgtgcaggatttgatctgttcaaatctaatgactgattttctttttttttttttccctcaggttactcgg ctgctgcctgatgcggttggtaccacttgtggtcagcggcttgagaaagtgtttggaacagagcattcacatattcttagggtcccctttaggaccgagaagggcatcgttcgcaaatggatctctcgctttgaagtctggccttacatggagacattcactgaggtgaagcaagctttctctattcatttttcaatcttccaatctgttttggcagcaat ttttcacttactaacactttggctttcgctaaaacttcggattttattacattaggatgtggcaaaagaaattgctgcagaactgcaggcaaagccagatcttataatcggcaactacagcgagggcaaccttgctgcctccttgttggctcacaagttaggtgtaactcaggtctgtaatgtttgtcacctgttatttcaactttgcatttccttt catttgcaactagaagttaagagttctctctcttttatcttttccgtctattttgccttctgcagtgcaccatagctcatgcgttggagaaaacaaaatatcctgattctgatatctacttgaagaaatttgatgaaaaataccatttctcagcccagtttactgccgatcttattgcaatgaatcacaccgatttcataatcaccagcactttccaggagatag cgggaaggtattacatcacaatggatttccgatatgattaaattagttaatttaatcctacttcattgtgtttgatcctaaaacttttctaaatttcccagcaaggacactgttggacagtacgagccacatggctttcacgatgcctggattgtatagagttgttcacggcattgatgtgttcgatcccaaattcaacattgtgtcacca ggagctgatatgaatctctatttcccctacttcgagaaggacagcatatcaccctgaaattgaggagctgctgtttagcgatgttgagaatgacgaacacatgtatgttactaaactagcaatcctgctgcaaaattgtggctaattatgtaaaaaagttttactgaatagatttgtgcttctatcaggtgtgtg ctgaaggacaggaataagccaattatattcaccatggctagattggatcgagtgaagaacttaactggacttgtggagttgtacgccaagaaccccggctaagggagttggttaaccttgtcgtggttggtggagaccgaaggaaggaatccaaagatttggaagaacaggcagagatgaagaagaagaatgtatgaacttaaaagacgcacaatttaaacggccaattccgat ggatttcttcccagatgaaccgcgtgaggaatggcgaactctacaggtacattgccgatactaggggagcttttgtgcagcctgcattttacgaggcttttggtttgactgttgttgaggccatgacctgtggtttgcctacgtttgcaactaatcacggtggtccagctgagatcatcgttcacgggaagtctggttttcacattga tccataccacggcgagcaggcagctgaacttctagctgatttctttgagagatgtaagaaagaaccttcacactgggaagccatttccgagggcggccttaagcgtatacaggagaagtaagcaagctgctactcttttcatttttgcaaaacctaccatgatcattattaagctcatttttgcaaaacctacttgttattctttgttgcttccttttccctgtttt ttgagccgaggttttatcgaaaacatgctttctaccttcacaaggtaggggtaaggtctgcgtttgttattattgttgttgttgattctgcgaattaattaattaaaaggtacacatggcaaatctactcggatcggttgttgacactggctgctgtttatggattctggaagcatgtttccaaacttgatcgtcttgaaattcgtcgttat cttgaaatgttctatgctctaaaattccgcaaactggtgagtttcactgctttctgcactcttccaattgttagttgagtgcactcatttaaactgtagctaaagctgttgtaaatcttcagttaagcagctgctaatgaagtttttatcttttgttttggttcaggctgaagctgtcccgttggctgttgagtaa

SEQ ID NO: 16: NtSUS4-T polypeptide sequence

MAERVLTRVHSLRERLDATLAAHRNEILLFLSRIESHGKGILKPHQLLAEFESIHKEDKNKLNDHAFEEVLKSTQEAIVLSPWVALAIRLRPGVWEYVRVNVNALIVEELTVPEYLQFKEELVNGTSNDNFVLELDFEPFTASFPKPTLTKSIGNGVEFLNRHLSAKMFHDKESMTPLLEFLRVHHYKGKTMMLNDRVQDLNTLQ NVLRKAEEYLTTLSPETSYSVFEHKFQEIGLERGWGDNAERVLEMICMLLDLLEAPDSCTLEKFLGRIPMVFNVVILSPHGYFAQENVLGYPDTGGQVVYILDQVPALEREMLKRIKEQGLDIKPRILIVTRLLPDAVGTTCGQRLEKVFGTEHSHILRVPFRTEKGIVRKWISRFEVWPYMETFTEDVAKEIAAELQAKPDLI IGNYSEGNLAASLLAHKLGVTQCTIAHALEKTKYPDSDIYLKKFDEKYHFSAQFTADLIAMNHTDFIITSTFQEIAGSKDTVGQYESHMAFTMPGLYRVVHGIDVFDPKFNIVSPGADMNLYFPYFEKEKRLTAYHPEIEELLFSDVENDEHMCVLKDRNKPIIFTMARLDRVKNLTGLVELYAKNPRLRELVNLVVGG DRRKESKDLEEQAEMKKMYELIKTHNLNGQFRWISSQMNRVRNGELYRYIADTRGAFVQPAFYEAFGLTVVEAMTCGLPTFATNHGGPEIIVHGKSGFHIDPYHGEQAAELLADFFERCKKEPSHWEAISEGGLKRIQEKYTWQIYSDRLLTLAAVYGFWKHVSKLDRLEIRRYLEMFYALKFRKLAEAVPLAVE

SEQ ID NO: 17: Polynucleotide sequence of NtSUS5-S

atggcctcaacagttgctgatagcatgcctgatgctttgaaacaaagccggtatcatatgaagagatgcttcgctaggtgaacacccttcttttgttttttcccctctacgtgtttatgtcaaatttccatgcataatgctaactacttttcttctttttgacttcaaaattggatgtgaaaaggttcattgcaatgggaaggaggc taatgaagttgaaacatttaacagaagaaatagaagaaactattgaagacaaggcagaaagaaccaggattttggagggttcacttggaaaaaattatgagttccacacaggtcagcaccatttaaccaacttagttgaacaggaaaaaagaaaaagcaaaagagttattgcaaggcgtaacgattttctttgaaattttcaggaggcagctgttgttccacc ttatgttgcttttgcagtaaggcacaatcctggcttctgggattatgtcaaagttaacgctgaaactctctctgtggaagctatttcagccagggaatattctcaaattcaaagagatgatctttgacgaagactggtaagtggaaaattgtatcattttaaagagaaacaattttgtaacatacaagaatagttttgatggttgaatgtgcaag cagggcaaaggatgataatgcactggaagtagattttggtgcttttgactactactctaatcctcggttagccctttcctcttctgtcggaaatgggctcaactttatctcaaaagttctgtcttcaaagtttggtggaaagccagaggacgcccagcctttgcttgattacttactagctcttaatcatcaaggagaggtatgaaaatggactacct ttgtttcttaaaggtattatataatgatgcgcgtttaaagttcctttttaaattgaaactttgcagaatctaatgatcaatgagaatctgaatggtgttgctaagcttcaagcagcattgatagtagctgaagtttttgtatcttcctttcccaaagacacaccttataaagactttgagcataagtaagcttctcatatgcttccattgtcatatgca gtataccaatgacatgctaccgaaaagttgtttgtttgtgacttgattatgaaaactctaggctcaaagaatggggctttgataaagggtggggtcacaatgcaggaagagtaagagagacaatgagactgctttccgagataatccaagcaccagatcccataaatatggagtcctttttcagcaagcttcctactacattcaacattgttatcttct ccattcatggttactttggccaagcagatgtccttggtctgcccgatactggaggccaggtctacatatacagcaatttatctccttttgcctcatattgcttattagcgacacttgcatcattgaaatcagacttttacttcacaggttgtttattctggatcaagtaagggctttgaggaggaaatgttacaaagaatcaagcagcaagggctaaacgtgaag cccaagattcttgtggtgagttttgcaaaaatatgcttagacaggttttgagattgatcggagaagggattaagatgatcaagatctttgtttcctgctttcatgatgtaaacaggtatctcgtctcataccagatgctcgagggacaacatgcaatcaggagatggaacctattcttaactcatcccattctcacatcctgagaattccattcaggactgaga aaggagttcttcgccaatgggtttctcggtttgatatctatccttacttggagaactatgccaaggcaagtcttctaacaaaattaccacctattcatacactttatttactttcttgaactaatcgttggttgtgacgtatatcattaggatgcttctgctaagatacttgagctcatggaaggtaaaccagacctcataattgggaactacactgat ggaaatttagtggcatctctattggccaacaaacttggagttactcaggttccgtagctgatcatatgatcatattttctacattgtttcttgataattaattaaatggaaatcttattggatgataacattttagggaaccattgctcatgcattagagaaaactaagtaagtatgaagattctgatgtgaagtggaagcagtttgatcccaagtaccacttttcttgccaattt actgccgatttattggcaatgaatgctgctgattttatcattaccagcacatatcaagaaatcgctggaaggttagcactgactctctcagtatatttggcaacttaatgaatttactgcagtggccaacactaaaagctatcattcgtccttcagcgaaactaggcctggacaatatgaaagtcacacagcatttaccatgccggggctttatagagctgtttca ggcatcaatgtatttgatccaaagttcaacattgctgctcctggggctgaacagtctacctatttccctttcactgagaaacagaaacgattcagcacatttcgtcctgctattaacgaattactttacagtaatgaggaaaacaatgagcacatgtaagtctaattgcccattttcctaatctaaccattgcttaaatcgttctgtttttaccggatgt gtggtacttatcagtaacatttttttttggatcagtggatttcttgcagaccggaaaaaaccaattatattttcaatggcgagatttgatacagtgaagaacctgtcaggcttgactgagtggtatgggaagaataagaagttgcggaacttggtaaaccttgttattgttgggggattcttcgatccatcaaaatcaaaagaccggggaggaag cagctgaaatcaagaagatgcatgaattgattgagaaataccagctcaagggacaaatgagatggatagcagctcaaactgataaatatcgaaatagtgagctataccgaactattgctgacactaagggagcttttgtccaaccggcttttatgaagcttttggactaaccgttattgaagcaatggattgtggattgcctacgtttgcaactaat caaggtggacctgcagaaatcattgttgatggggtttcaggtttccatattgatccttacaatggggacgaatcaagcaagaaaatagctgatttctttgagaagtgtaaggttgattctaaatttggaacaggatatctgagggaggtctcaagcgcattgaagaatggtaacaaactagttccaagtttaaaaaatggaaaaaatgcttatcat gttatattttcgtggttttaagttctgcttcgatgcagttatacgtggaagatttatgcaaacaaagtgttgaatatgggatcaatctatggattttggagacaattcaatgtggggcaaaagcaggctaagcaaagatactttgagatgttttacaatcctctcttcaggaaattggtaggttgtatatgttgaatacaatttactaaga tcctcaaaatgaccaagaaatatacattgactatgctacttttgtaatttcacaggccaaaagcgtgccgatcccacatgaagagccattgccacttgcaacatcagactctactcaatcccaagaattaaaactaccactaccagttccagcagcagtagctaaagttctgccattaacaaggcatgcttttaacttaattacttctctacctagagtaactggtaaagtggat gtcaagtga

SEQ ID NO: 18: NtSUS5-S polypeptide sequence

MASTVADSMPDALKQSRYHMKRCFARFIAMGRRLMKLKHLTEEIEETIEDKAERTRILEGSLGKIMSSTQEAAVVPPYVAFAVRHNPGFWDYVKVNAETLSVEAISAREYLKFKEMIFDEDWAKDDNALEVDFGAFDYSNPRLALSSSVGNGLNFISKVLSSKFGGKPEDAQPLLDYLLALNHQGENLMINENLNGVAKLQAALI VAEVFVSSFPKDTPYKDFEHKLKEWGFDKGWGHNAGRVRETMRLLSEIIQAPDPINMESFFSKLPTTFNIVIFSIHGYFGQADVLGLPDTGGQVVYILDQVRALEEEMLQRIKQQGLNVKPKILVVSRLIPDARGTTCNQEMEPILNSSHSHILRIPFRTEKGVLRQWDASAKILELMEGKPDLIIGNYTDGNLVA SLLANKLGVTQGTIAHALEKTKYEDSDVKWKQFDPKYHFSCQFTADLLAMNAADFIITSTYQEIAGSETRPGQYESHTAFTMPGLYRAVSGINVFDPKFNIAAPGAEQSTYFPFTEKQKRFSTRPAINELLYSNEENNEHILADRKKPIIFSMARFDTVKNLSGLTEWYGKNKKLRNLVNLVIVGGFFDPSKSKDREEAAEI KKMHELIEKYQLKGQMRWIAAQTDKYRNSELYRTIADTKGAFVQPALYEAFGLTVIEAMDCGLPTFATNQGGPAEIIVDGVSGFHIDPYNGDESSKKIADFFEKCKVDSKYWNRISEGGLKRIEECYTWKIYANKVLNMGSIYGFWRQFNVGQKQAKQRYFEMFYNPLFRKLAKSVPIPHEEPLPLATSDSTQSQEL KLPLPVPAAVAKVLPLTRHAFNLITSLPRVTGKVDVK

SEQ ID NO: 19: Polynucleotide sequence of NtSUS5-T

atggcctcaactgttgctggtagcatgcctgctgctttgaaacaaagccgatatcatatgaagagatgcttcgctaggtgaacacccttcttgttctttttgttttttccctctaccatttatgtcaaatttcaatgcataatgctaactactttttttctttttgacttcaaaattggacgtgaaaaggttcattgcaatgggaaggaggttgat gaagctgaaacatttaacagaagaaatagaaaaaactattgaagacaaggcagaaagaaccaagattttggagggttcacttggaaaaaattatgagttccacacaggtcagcaccatttaaccaacttaattgaataggaagaaaaaaaaaagcaaaagagttattgcaaggcgtaacgatttcctttgaaattttcaggaggcagctgttgtc ccaccttatgttgcttttgcagtaaggcacaatcctggcttctgggattatgtcaaagttgacgctgaaactctctctgtggaagctatttcagccagggactatctcaaattcaaagagatgatctttgaattggtaactggaagattgtatcattttaaagaaacaattttttaatattcaagattagttttgatggttgaatgtgcaagcaggg caaaggatgaaaatgcactcgaagtagattttggtgcttttgactactactctaatcatcggttagccctttcctcttctgtcggaaatgggctaaacttcatctcgaaagttttgtcttcaaagtttggtggaaaaggcagaagaatgcccagcctttgcttgattacttactagctcttaatcatcaaggagaggtatggaaatggactacctt cctttcttaaggaattatataatgatgtatgttataaagatcctttttaaacattgacactttgcagaatctaatgatcaatgagaatctgaatggcgtctctaagcttcaagcagcattgatagtagtagctgaagttttgtatcttcctttcccaaagacacaccttataaagactttgagcataagtaagcttttcaaacgcttctgttatcatatgcaatata ccaagaatatgttgccttttgaaaagttgtttgtttatgacttgataatgaaaatactaggctcaaagaatggggctttgagaaagggtggggtcacaatgcaggaagagtaagagagacaatgagactgctttccgagataatccaagcgccagatcccataaatatggagtcctttttcagcaggcttcctactacattcaacattgttatctctcca ttcatggttactttggccaagcagatgtccttggtttgcccgatactggaggccaggtttacatacacagcaatttatctccttttgcctcatatttacttattagcgacacttgcattattgaaatcacatttgtatttaacaggttgtttatattctggatcaagtaagagccttagaggaggaaatgttacaaagaatcaagcagcaagggttaaatgtgaagcccaaga ttcttgtggtgagttatgcaaaaatatgcgtagccaaggttttgaaattgttcagaggggattaagatgatcgagatatttgtttccttcttccattgatgtgtacaggtcactcgtctcattccagatgctcgagggactacatgcaatcaggagatggaacctatacttaactcgtcccattctcacatcctgagaattccattcaggacagaga aaggagttcttcgccaatgggtttctcggtttgatatctatccttacttggagaactatgccaaggcaagtctcctaccaaaattaccacctattcatacacttattcagttttttgagctaatcattctcatttgtcacgtatgtgattaggatgcttctgctaagatacttgagctcatggaaggtaaaccagacctcattattgggaactacactgatgga aatttagtggcatctctattggccaacaaacttggagttactcaggttctacagctgatcatttatctgatcagattttctacattgttttcttgataattaattaaacggaaatcttatgagattgtaacattttagggaaccattgctcatgcattagagaaaaccaagtatgaagattctgatgtcaagtggaagcagtttgattccaagtaccacttttcttgccaatt cactgccgatttattggcaatgaatgctgctgattttcattaccagcacatatcaagaaatcgcaggaaggttagcactgactctctcagtatatttggcaacttaatgaatgtactgcttgtggccaacactaaaagctattactcgtccttcagcgaaactaggcctggacaatatgaaagtcacacagcatttaccatgccggggctttatagagctgtt tcaggcatcaatgtatttgatccaaagttcaacattgctgctcctggggctgaacagtctgcctatttccccttcactgagaaacagaaacgattcagcgcgtttcgtcctgctattgaggaactactttacagtaatgagcaaaacaacgagcacatgtaagtctaattgccccattttcctaatctaaccattgcttaaatgttctgtt tttacttgatatgtggtacttatcagtgatattttttattggaacagtggatttctgcagaccgtaaaaaaccaattatattttcaatggcaagatttgatacggtgaagaacttgtcaggcttgactgagtggtatgggaagaataagaagttgcggaacttggttaacctcgttatcgttgggggattcttcgatccatcaaaatcaaa agaccgggaggaagcagctgaaatcaagaagatgcatgaattgattgagaaatacaagctcaagggacaaatgagatggatagcagctcaaactgataaatatcaaaacagtgagctatatcgaactattgctgacactaaaggagctttcgtccaaccggctttatgaagcttttggactaactgttattgaagcaatgaattgtggactgcctacattt gctactaatcaaggcggacctgcagaaatcattgttgatggggtttcaggcttccatattgatccttacaatggggatgaatcgagcaagaaaatagctgatttctttgagaagtgtaaggttgattctaaatattggaacaagatatgtggaggaggtctcaagcgcattgaagaatggtaa

SEQ ID NO: 20: NtSUS5-T polypeptide sequence

MASTVAGSMPDALKQSRYHMKRCFARFIAMGRRLMKLKHLTEEIEKTIEDKAERTKILEGSLGKIMSSTQEAAVVPPYVAFAVRHNPGFWDYVKVDAETLSVEAISARDYLKFKEMIFDEDWAKDENALEVDFGAFDYSNHRLALSSSVGNGLNFISKVLSSKFGGKAEDAQPLLDYLLALNHQGENLMINENLNGVSKLQAALI VAEVFVSSFPKDTPYKDFEHKLKEWGFEKGWGHNAGRVRETMRLLSEIIQAPDPINMESFFSRLPTTFNIVIFSIHGYFGQADVLGLPDTGGQVVYILDQVRALEEEMLQRIKQQGLNVKPKILVVTRLIPDARGTTCNQEMEPILNSSHSHILRIPFRTEKGVLRQWDASAKILELMEGKPDLIIGNYTDGNLVASL LANKLGVTQGTIAHALEKTKYEDSDVKWKQFDSKYHFSCQFTADLLAMNAADFIITSTYQEIAGSETRPGQYESHTAFTMPGLYRAVSGINVFDPKFNIAAPGAEQSAYFPFTEKQKRFSAFRPAIEELLYSNEQNNEHIGFLADRKKPIIFSMARFDTVKNLSGLTEWYGKNKKLRNLVNLVIVGGFFDPSKSKDREEAAEI KKMHELIEKYKLKGQMRWIAAQTDKYQNSELYRTIADTKGAFVQPALYEAFGLTVIEAMNCGLPTFATNQGGPAEIIVDGVSGFHIDPYNGDESSKKIADFFEKCKVDSKYWNKICGGGLKRIEEW

SEQ ID NO: 21: Polynucleotide sequence of NtSUS6-S

atggctactgcaccagccctaaatagatcagagtccatagctgatagcatgccagaggccttaaggcaaagccggtaccacatgaagaaatgttttgccaagtacatagagcaaggaaagaggatgatgaaacttcataacttgatggatgagttggagaaagtaattgatgatcctgctgaaaggaaccatgttttggaaggcttacttggctacatattatgcactac aatggtatagctagattcatatgtacttatgatgcccttatattgtttcctgatgtattacttaaaaccttcttttgatcaaatttacaggaggctgcagttgttcctccctacattgcctttgccacgagacagaatcctggattctgggaatatgtgaaagtgaatgctaatgatctttctgttgagggtattacagctacagaatacttgaa attcaaggaaatgatagttgatgaatgctggtatagtatacgttgcagcttatcataccttttgtggttttataacttcaatcagaaaactcatcagagttacctttgtgtgaacatgaaatgcagggcaaaagatgaatatgcactggaaattgattttggagcagtagacttctcaacgcctcgactgaccctatcctcttcaattggcaatggt ctcagttatgtttccaagtttctaacttcaaagctaaatgctacctccgcgagtgcacagtgtctggttgactacttgctcactttgaatcatcaaggagatgtacgtcaacaaaaatcaaactccataagtaaacttgtcaactctaagaagaaaaaataggaaaagaagattcacgtaacaaattttctttatgttcaactgcagaa actgatgatcaatgagacactcagcactgtctcaaagcttcaggctgcactggttgtagcagaagcatctatttcctctttaccaacagatacaccatatgagagctttgagctaaggtgatttgttttttcctctacttccctccacttgtgccatgctacgtagtactaagtaacttcaattcttgtaaagattcaaacagtggggttttgagaaagga tggggtgatacagctgaaagggtcagcgacaccatgagaacactgtctgaggtgcttcaggcaccagatccattgaacattcagaagttctttggaagggttccaactgttttcaatattgtattgttctctgtccatggatactttggccaagcagatgttcttggcttgccagacactggtggtcaggtaagcatttaatagcttttacatttaacttctatgcat tgacaataaaataatttttaacagtttgaccacttctgctcttgttcaacaggtagtttatgttttggatcaagttgtagcttttgaagaagaaatgctacaaagaattaaacagcaggggctcaatattaagcctcaaattcttgtggtgagttcctgacaatcgacgtgactatgcaattatgtagaggctgttagaaaagttaatatcatatgttga ttgcacagttaacccgactgattccggatgcaaaaggaacaaagtgcaaccaggaactagaaccaatcaagaatacaaaacattcacacatcctcagagttccatttaggacagaaaaaggagtgcttaatcaatgggtttcacgatttgatatctatccatatctggagagatatactcaggtatgtatttttatatcaaccttgctcatcaaagatgt gttgtttcctcaattccatttttccccttggcaaaaggatgctgctgacaaaatcgtcgagctaatggaaggcaaacctgatctaatcattggtaactacactgatgggaatctagtggcttcactaatggctagaaaacttgggataactctggtaacttttcttaatcatatttgatgttgcttcttctccaagttagttcttaatctccactgacc tagaccatctttgcaacagggaactattgctcatgctttggagaagacaaaatattgaagactctgacataaaattgaaggaactcgatccgaagtaccacttctcttgccaattcacagctgatttgattgcaatgaattcagcagatttcattatcactagcacataccaagaaaatagctggaaggtaagaattagagctaataagtaatgcattcatatgtatttcagcat cgctctttcaccatcatcgaatacacaccactactcagtaaatgtatttgctcaaaagtttgcaacttaatggatctcattcttgaatgcttcaacatatgcagcaaagataaaccaggacagtgcatttacccttccaggggctttacagagttgcttcaggtatcaatgtctttgatccaaaatttaatattgctgcacctggggca gaccagtcggtgtatttcccttacacagaaaagcagaagcgtttgactgctttccgccctgccattgaggaactgctttttagtaaagtggacaatgacgagcacgtgtaagtctaagtgttaaacttcagcttagtgcctagaacatcccactgctctatgtattgatgtttcacttgtttcaaacagtggatatttagaagacaga aagaaacctatcctgtttaccatggcaaggctggacacagtgaagaacacatctggactaacagaatggtatggcaagaagaacaagaggctcagaagcttagttaaccttgttgtggttggtggttcctttgatcctacaaaatccaaggataggggaagaagcagctgaaataaaaaagatgcacatgctgatagagaaataccagcttaagggtcagattagatggatagca gctcagactgacagatacagaaatagtgaactctaccgcacaatagcagattccaaaggagcttttgtgcagcctgcattgtatgaagcatttggtctaacagtcattgaggcaatgaactgtggattaccaacctttgctaccaaccaaggtggccctgctgagattattgttgatggggtctcaggctttcatattgatccaaataatggggatgaatcaagca acaaaattgccaactttttccaaaaatgcagggaggatcctgagtattggaacaggatttcagtccagggtctaaaccgtatatatgaatggtaactcacagataagccattcaaattgcaaagaggcacatatcttgcagaaaatttcttaatccttaaatcctaattttttgcagttacacatggaagatctatgcaaacaaggtattgaat atggggtccatctatactttttggaggacattgtacagagatcagaaacaagcaaagcaaagatacatcgagactttctacaatcttgagtttaggaacttggtatagtgctgcatgacattgacagtataccacaaacatctttatgagatgaattacttttaataaaattgtttttaacctttgcttccttaatggcacttattgcaggtaaaaaatgtgcc tatcagaaaggacgaaacaccacaaggaccaaaggagggagaaagttaagccacagatatcacaaaggcatgctctaaagcttttgcctacagtttttcaagagaccctagtatattctagtactaaattagaattatacagcatgcagctttgctgttcacctttctaaatcaccagttgtgtgtcaatcaagttgacaaaatcaataaattgggatt ttccctttcctatgcttgattgttattactcctactttgtttatggtagtcttccttcattgttttctcctgtacttcttttactacaactgtactgacatactaattatttctgtgtaccaggcgctcacaatcaaggttgcagaagtaagattagataaaattgctactgcatga

SEQ ID NO: 22: NtSUS6-S polypeptide sequence

MATAPALNRSESIADSMPEALRQSRYHMKKCFAKYIEQGKRMMKLHNLMDELEKVIDDPAERNHVLEGLLGYILCTTMEAAVVPPYIAFATRQNPGFWEYVKVNANDLSVEGITATEYLKFKEMIVDECWAKDEYALEIDFGAVDFSTPRLTLSSsignGLSYVSKFLTSKLNATSASAQCLVDYLLTLNHQGDKL MINETLSTVSKLQAALVVAEASISSLPTDTPYESFELRFKQWGFEKGWGDTAERVSDTMRTLSEVLQAPDPLNIQKFFGRVPTVFNIVLFSVHGYFGQADVLGLPDTGGQVVYVLDQVVAFEEEMLQRIKQQGLNIKPQILVLTRLIPDAKGTKCNQELEPIKNTKHSHILRVPFRTEKGVLNQWRFDIYP YLERYTQDAADKIVELMEGKPDLIIGNYTDGNLVASLMARKLGITLGTIAHALEKTKYEDSDIKLKELDPKYHFSCQFTADLIAMNSADFIITSTYQEIAGSKDKPGQYESHSAFTLPGLYRVASGINVFDPKFNIAAPGADQSVYFPYTEKQKRLTAFRPAIEELLFSKVDNDEHVGYLEDRKKPILFTMARLDTVKNTSGLTEWY GKNKRLRSLVNLVVVGGSFDPTKSKDREEAAEIKKMHMLIEKYQLKGQIRWIAAQTDRYRNSELYRTIADSKGAFVQPALYEAFGLTVIEAMNCGLPTFATNQGGPAEIIVDGVSGFHIIDPNNGDESSNKIANFFQKCREDPEYWNRISVQGLNRIYECYTWKIYANKVLNMGSIYTFWRTLYRDQKQAKQRYIE TFYNLEFRNLVKNVPIRKDETPQGPKEREKVKPQISQRHALKLLPTVFQETLALTIKVAEVRLDKIATA

SEQ ID NO: 23: Polynucleotide sequence of NtSUS6-T

atggctactgcaccagccctgaaagatcagagtccatagctgatagcatgccagaggccttaaggcaaagccggtaccacatgaagaaatgttttgccaagtacatagagcaaggcaagaggatgatgaaacttcataacttgatggatgaattggagaaagtaattgatgatcctgctgaaaggaaccatgttttggaaggcttacttggctacatattatgtactac aatggtatagctagattcatatgtacttatgatgtccttatattgtttccgggaggcattattcttaaatccttctttgatcaaatttgtaggaggctgcagttgttcctccctatattgccttcgccacgagacagaatcctggattctgggaatatgtgaaagtcaatgctaatgatctttctgttgagggtattacagctacagattacttgaaattca aggaaatgatagttgatgaaagctggtatagaatactttgcagcttatcataccttttgtggttttataatttcaatcagaaaactcatcagagttacctttgtgtgaacatgacatgcagggcaaaagatgaatatgcactggaaattgattttggagcagtagacttctcaacgcctcgactgaccctatcctcttcaattggaaatggtctcagtt atgtttccaagtttctaacttcaaagctaaatgctacctcaaagctaaatgctacctcagcgagtgcacagtgtctggttgactacttgctcactttgaatcaccaaggagatgtacgtcaacaaaaatcaaactccataagtaaacttgtcaactctaagaagtaaaaataggaaaagaagattcatgtaacaaattttctttatgttcaactgtagaaactgatgat caatgagacactcggcactgtctcaaagcttcaggctgcactggttgtagcagaagagcatctatttcctccttaccaacagatacaccataccagagctttgagctaaggtgatttgttttttcctctacttccttccacttttggtgtgctacatagtactaagtaacttcaattcttgtaaagattcaaacagtggggttttgagaaaggatggggtgat acagctgaaagggtccgcgacaccatgagaacactttctgaggtacttcaggcgccagatccattgaacattgagaagttctttgggagggttccaactgttttcaatattgtattgttctctgttcatggatactttggccaagcaaatgttcttggcttgccagacacaggtggtcaggtaagcatctaatagcttttacatttaacttctatgcattgacaataaaataact tctacactaccaaataatttttgaaagtttgaccacttcggctcttgttcaacaggtggtttatgttttggatcaagttgtagcttttgaagaagaaatgctccaaagaattaaacagcaggggctcaatattaagcctcaaattcttgtggtgagctcctagacaatgacgtgactatgcaattaagtagaggctgttagaaaagttaatatcatatgttgatt gcacagttaacccgactgattccggacgccaaaggaacaaagtgcaaccaggaactagaaccaatcaagaatacaaaacattcacacatcctcagagttccatttaggacagaaaaaggagtgcttaatcaatgggtttcacgatttgatatctatccatatctggagagatatactcaggtgtgtatttttatatcaaccctgctcatcaaagatgtg ttgtttcctcaattccatttttcgccttgacaaaaggacgctgctgacaaaatcatcgagctaatggaaaggcaaacctgatctaatcattggtaactacactgatgggaatctagtggcttctctaatggctagaaagcttgggataactctggtaacttttcttatcatatttgatgttgtttcttctccaagttggttcttaatgtcaactaaccca gaccatctttgtaacagggaactattgctcatgctctggagaagacaaaatatgaagactctgacatcaaattgaaggaactcgatccgaagtaccacttttcttgccaattcacagctgatttgattgcaatgaattcagcagatttcattatcacaagcacatatcaagaaaatagccggaaaggtaagaattggaactacggaagcagagagctaataagtagtgcact catatatttcagcatcgctctttcgcataatcgaatacacaccactactcagtaaatgtacttgctcaaaagtttacaagtttatggatcttattcttgaatgcttcaacatatgcagcaaagataggccaggacagtatgagagccatagtgcatttacccttccagggctttacagagttgcttcaggcatcaatgtctttgatcctaaatttaatttgctg cacctggggcagaccaatcggtgtatttcccttacacagaaaagcagacgcgtttgactgctttccgccctgccattgaggaactgctttttagtaaagtggacaatgacgagcacatgtaagtcttagtgttaaacttcagctttcagcttagtgcctagaacattccactggctctatgtattaatgtttcacttgtttcaaacacagtgg atatttagaagacagaaagaaacctatcctgtttaccatggcaaggctggacacagtgaagaacacatctggactaacagaatggtatggcaagaacaagaggctcagaagcttagttaaccttgttgtggttggtggttcctttgatcctacaaaatccaaggatagagaagaagcagctgaaataaaaaagatgcacatgctgatagagaaataccagcttaagggtcagat cagatggatagcagctcagactgacagatatagaaacagtgaactctaccgcacaatagcagattccaaaggagcttttgtgcagcctgcattatatgaagcatttggtctaacagtcattgaggcaatgaactgtggattaccaacctttgctaccaaccaaggtggccctgctgagattattgttgatggggtctcaggctttcatattgatccaaataatgggg atgaatcaagcaacaaagttgccaactttttccaaaaatgcagggaggatcctgagtattggaacaggatttcagtccagggtctaaaccgtatatatgaatggtaactcacagataagccattcaaattgcaaagaggcacatatcttgctgaaaatttcttaatcctttaatcctaaaattttgcagttacacatggaagatctatgcaaacaa ggtattgaatatggggtccatctatactttttggaggacattgtacagagatcagaaaaagcaaagcaaagatacatcgagactttctacaatcttgagtttaggaacttggtatagtgctgcatgacattgacagtataccacaaacatcttttgagatgaattacttttaataaaattgtttttaacctttgcctccttaatgacacttattgcaggtaaaaa atgtgcctatcagacaggacgaaacaccacaaggaccaaaggagaggagggagaaagttaagccacagatatcacaaaggcatgctctaaagcttttgcctatagtttttcaggagaccctagtatattctagtactaaattagaattatacagcatgcagcttgcttctgctgttcacctttctaaatcaccagttatgtcaatcaagttgacaaaatcaata aattcggcttttccctttcctatgcttgattgttattactcctacttcgtttatggtagtcttccttcattgttttctcctgtacttcttttactacaactgtactga

SEQ ID NO: 24: NtSUS6-T polypeptide sequence

MATAPALKRSESIADSMPEALRQSRYHMKKCFAKYIEQGKRMMKLHNLMDELEKVIDDPAERNHVLEGLLGYILCTTMEAAVVPPYIAFATRQNPGFWEYVKVNANDLSVEGITATDYLKFKEMIVDESWAKDEYALEIDFGAVDFSTPRLTLSSsignGLSYVSKFLTSKLNATSASAQCLVDYLLTLNHQGDKLM INETLGTVSKLQAALVVAEASISSLPTDTPYQSFELRFKQWGFEKGWGDTAERVRDTMRTLSEVLQAPDPLNIEKFFGRVPTVFNIVLFSVHGYFGQANVLGLPDTGGQVVYVLDQVVAFEEEMLQRIKQQGLNIKPQILVLTRLIPDAKGTKCNQELEPIKNTKHSHILRVPFRTEKGVLNQWVSRFDIYPY LERYTQDAADKIIELMEGKPDLIIGNYTDGNLVASLMARKLGITLGTIAHALEKTKYEDSDIKLKELDPKYHFSCQFTADLIAMNSADFIITSTYQEIAGSKDRPGQYESHSAFTLPGLYRVASGINVFDPKFNIAAPGADQSVYFPYTEKQTRLTAFRPAIEELLFSKVDNDEHIGYLEDRKKPILFTMARLDTVKNTSGLTEWYGKNK RLRSLVNLVVVGGSFDPTSKSKDREEAAEIKKMHMLIEKYQLKGQIRWIAAQTDRYRNSELYRTIADSKGAFVQPALYEAFGLTVIEAMNCGLPTFATNQGGPAEIIVDGVSGFHIDPNNGDESSNKVANFFQKCREDPEYWNRISVQGLNRIYECYTWKIYANKVLNMGSIYTFWRTLYRDQKQAKQRYIETFYN LEFRNLVKNVPIRQDETPQGPKERREKVKPQISQRHALKLLPIVFQETLVYSSTKLELYSMQLASAVHLSKSPVMSIKLTKSINSAFPFPMLDCYYSYFVYGSLPSLFSPVLLLQLY

--->

SEQUENCE LIST

<110> Philip Morris Products S.A.

<120> MODULATION OF THE CONTENT OF REDUCING SUGAR IN THE PLANT

<130> P10505EP

<140>EP18164769.4

<141> 2018-03-28

<160> 24

<170> PatentIn version 3.5

<210> 1

<211> 4384

<212> DNADNA

<213> Nicotiana tabacum

<220>

<221> other_characteristic

<222> (1520)..(1520)

<223> n is a, c, g, or t

<400> 1

atggcagcta gtggtcttag cattaagaaa agtttggagg aatccatttt ggctcatcca 60

gatgaaattt tggctctcaa gtcaaggtac attactacat ataatgatat taagaactag 120

aggcttatcc aaggttttgt tacatttttg aaattataag tttagaacct aatagtactt 180

ggtagcactt gtttccttat tatctagctg ttgttactgc ttgttgctac tgctttctgt 240

tcatctttcc ttgagcccgg tctatcggaa acaacctctc tattctcaaa gtataaggtt 300

tgcgtacata ctacctcccc agactctact tgtggaattt actgtttttg ttgtgttgtt 360

gtaatctaat atttattaga attttactga tttttcacat atatatatct atgtcccctg 420

tcgaaaattc tatagctcat gttagctaaa tacattagta ccattgtttt taattgtttt 480

ggttttggca caggattgaa actgaaggga aaggggtaat gaaaccactt gatctcttga 540

accatttggt ttctgttact agtaagacaa atggagtaaa tattgtacct agtgcacttg 600

tggaagttct cagttgcagc caagaagctg tgattgtacc accaaaacta gcactagctg 660

tacgtccgag gcccggtgta tgggagtact tgtcactgaa tcttaagaca aagaaagtgg 720

ctgaattaag cattcctgaa taccttcaat tgaaagagaa cactgttgat gaaaggtaaa 780

gtattagtct gcgatttcgc tttgtgaaat tgaagttttt gttttgattc ataatgtttt 840

gtgtatcaat tatgttacca gtggaaacat attggagttg gattttgagc catttacaac 900

agttacacca ccaaaaacac tttctgactc tattggcaat ggtttggagt ttcttaatcg 960

ccacattgct tcgaaaatgt ttcatgataa ggagatttcc agatgcctcc ttgacttcct 1020

cagaaaccat aactacaaag gaaaggtaat aaaaaaaagt gtttctttaa acaagttgta 1080

1140

gcattcaaag cctagagagt ttccaacttg ttctgaaaaa agcagaggaa catttgtgca 1200

cattgaatcc agaaactcca tactccaatt ttgaatcaaa gtttgaagag attggcttgg 1260

aaagagggtg gggaaacacc gctgaacgcg tgcaagacac tatcagtcat cttttgcatc 1320

tccttgaggc tcctaacgcg tcttctttgg aaaatttcct tggtagaatc ccattggttt 1380

tcaatgttgt gattctaact ccacatggtt attttgctca agataatgtc ttgggctatc 1440

ctgacactgg tggccaggtt tgtgtccaat attttgcatt cttgatcaag ttctttatac 1500

catttgaacc aacaatcttn aacattcttt ttttggttgt gaaatgttga ataggttgtt 1560

tacattcttg atcaagttcc agctatggag cgtgagatgc ttcatcgtat gaagcttcaa 1620

ggactcgatg atatcatccc tcgcatcctt gttgtaagtg gccttaattt tcctagtttc 1680

atttacacct ctaaatgaaa ttgatctttt ttgttgtttt atatcaggta acaaggctgc 1740

tgcctgatgc agtaggaacc acctgtggcg agcggatgga gaaagtatat ggggcagaac 1800

attctcatat aattcgtgtt ccatttagaa ctgagaaggg aatgttgcgc aaatggatct 1860

cacgattcga agtctggcca tacatggaaa ctttcactga ggttggaaca taaaaacaaa 1920

taaaatccat tggaatgttc cttctgcaat tgaaaatgtc ttgctaactg aagacccatt 1980

tttaaattga tcatcaggat gttgcagaag aacttgtcaa agaattgcaa gctaaaccag 2040

acttgatcat tggaaactac agtgagggaa atcttgctgc ctctttgctt gcgaagaaat 2100

ttggggctac tcagtgtact attgctcatg ccttggaaaa aactaagtat ccaaactctg 2160

accttaattg gaagaagttt gatgacaagt atcatttctc aagtcagttc actgctgatc 2220

tctttgccat gaatcacact gatttcatca tcaccagcac tttccaagaa attgctggaa 2280

ggtaaaagca aatgcacacc atcatagtat ttcatattttt tacccttgtt tatactattt 2340

ccattcaccg accccgactt gtttaggatt gagccatagt tgttgttgtt gtttgtttat 2400

actatttcca tttgccgacc acaacttgtt taggactgag gtatagttgt tgttgttggt 2460

ttgttcatat tattttcatt cgctaaccct aacttgtttg ggactgaggc atagtagtag 2520

tagtagttgt tgctattagt ttatactatt tccatttgcc aaccccaact tgtttggtac 2580

tgagacatag ttgttgttgt tgttgtttgt ttatactatt tccatttgcc gaccccaact 2640

tgtttaggac tgaggtatag ttgttgttgt tggtttgttc atattatttt cattcgctaa 2700

ccccaacttg tttgggactg aggcatagta gtagtagtag tagttgttgc tattagttta 2760

tactatttcc atttgccaac cccaacttgt ttggtactga gacatagttg ttgttgttgt 2820

ttgtttatac tatttcaatt tgtcgacccc aatttgtttg ggaccaaggc atggttgttg 2880

ttgttgtttg tttgttttta ctgtttccat tgatattgga acatttgtta tttgcagcaa 2940

aaacactgta ggacagtatg agagtcatac tgcttttacc atgcctggat tgtaccgagt 3000

agtccatgga atcgattcgt ttgatccaaa gttcaacatt gtctcccctg gggctgatat 3060

gtcaatctac ttcccttaca ctgagaagga gaaaaggcta accaacttcc acccggaaat 3120

3180

3240

tcaacataaa cagatgtgtg ttgaaggacc ggaacaagcc aattctcttt accatggcaa 3300

ggctagatcg cgtgaagaat ctaacagggc tcgtggaatg gtatgctaag aatgcaaggc 3360

tgagggagct tgttaacctt gtggttgtag gcggagacag aaggaaagaa tccaaagatt 3420

tagagagca agcagagatg aagaagatgt atgatcttat cgaaacctat aacctgaacg 3480

gccaattcag gtggatttct tcccaaatga atcgtgtgag gaacggagaa ctctatcgtt 3540

acattgcaga cacgaggggt gctttcgttc aaccagcatt ctacgaggct tttggtttga 3600

3660

ttgagattat agtgaatgga aaatctggtt tccatattga tcctaatcaa ggtgacaagg 3720

ctgctgatat gttggtaaat ttctttgaaa aatctaaaga agatccaagt tattgggatg 3780

ctatttccaa gggaggtctg caacgtattc ttgaaaagta agcttttgca tttgattagc 3840

acaagtgcac aaccaagatt taacttttga acaaactaaa actaaccctt ttttgtattt 3900

tcttttgcta ggtatacatg gcaaatttat tcacagaaag tgatcacact atctgggatt 3960

tatggattct ggaagtatgc aaccaagaat gataaagttg ctagtgcaaa gaagcgctat 4020

4080

ccttggatca acggttaagt tgtctttgtg caacctatag gtcaggggtt tgagccgtag 4140

aagtagccac taatatttac attagggtag actgtgtaca tatcacaccc cttggggtac 4200

ggccctttcc tggatcctgt atgaacgcgg gatgccttgt gcaccggggct gtattttttt 4260

ttttagtgtc acttctgtat tttgtttgag cttgtttata aagtttggaa atctgctgct 4320

aatttgtata tttgttggtt gtgtatttca ggctgagaaa gttccattgg ctattgatga 4380

atag 4384

<210> 2

<211> 803

<212> PROTEIN

<213> Nicotiana tabacum

<400> 2

Met Ala Ala Ser Gly Leu Ser Ile Lys Lys Ser Leu Glu Glu Ser Ile

1 5 10 15

Leu Ala His Pro Asp Glu Ile Leu Ala Leu Lys Ser Arg Ile Glu Thr

20 25 30

Glu Gly Lys Gly Val Met Lys Pro Leu Asp Leu Leu Asn His Leu Val

35 40 45

Ser Val Thr Ser Lys Thr Asn Gly Val Asn Ile Val Pro Ser Ala Leu

50 55 60

Val Glu Val Leu Ser Cys Ser Gln Glu Ala Val Ile Val Pro Pro Lys

65 70 75 80

Leu Ala Leu Ala Val Arg Pro Arg Pro Gly Val Trp Glu Tyr Leu Ser

85 90 95

Leu Asn Leu Lys Thr Lys Lys Val Ala Glu Leu Ser Ile Pro Glu Tyr

100 105 110

Leu Gln Leu Lys Glu Asn Thr Val Asp Glu Ser Gly Asn Ile Leu Glu

115 120 125

Leu Asp Phe Glu Pro Phe Thr Thr Val Thr Pro Pro Lys Thr Leu Ser

130 135 140

Asp Ser Ile Gly Asn Gly Leu Glu Phe Leu Asn Arg His Ile Ala Ser

145 150 155 160

Lys Met Phe His Asp Lys Glu Ile Ser Arg Cys Leu Leu Asp Phe Leu

165 170 175

Arg Asn His Asn Tyr Lys Gly Lys Ser Leu Met Val Lys Glu Ser Ile

180 185 190

Gln Ser Leu Glu Ser Phe Gln Leu Val Leu Lys Lys Ala Glu Glu His

195 200 205

Leu Cys Thr Leu Asn Pro Glu Thr Pro Tyr Ser Asn Phe Glu Ser Lys

210 215 220

Phe Glu Glu Ile Gly Leu Glu Arg Gly Trp Gly Asn Thr Ala Glu Arg

225 230 235 240

Val Gln Asp Thr Ile Ser His Leu Leu His Leu Leu Glu Ala Pro Asn

245 250 255

Ala Ser Ser Leu Glu Asn Phe Leu Gly Arg Ile Pro Leu Val Phe Asn

260 265 270

Val Val Ile Leu Thr Pro His Gly Tyr Phe Ala Gln Asp Asn Val Leu

275 280 285

Gly Tyr Pro Asp Thr Gly Gly Gln Val Val Tyr Ile Leu Asp Gln Val

290 295 300

Pro Ala Met Glu Arg Glu Met Leu His Arg Met Lys Leu Gln Gly Leu

305 310 315 320

Asp Asp Ile Ile Pro Arg Ile Leu Val Val Thr Arg Leu Leu Pro Asp

325 330 335

Ala Val Gly Thr Thr Cys Gly Glu Arg Met Glu Lys Val Tyr Gly Ala

340 345 350

Glu His Ser His Ile Ile Arg Val Pro Phe Arg Thr Glu Lys Gly Met

355 360 365

Leu Arg Lys Trp Ile Ser Arg Phe Glu Val Trp Pro Tyr Met Glu Thr

370 375 380

Phe Thr Glu Asp Val Ala Glu Glu Leu Val Lys Glu Leu Gln Ala Lys

385 390 395 400

Pro Asp Leu Ile Ile Gly Asn Tyr Ser Glu Gly Asn Leu Ala Ala Ser

405 410 415

Leu Leu Ala Lys Lys Phe Gly Ala Thr Gln Cys Thr Ile Ala His Ala

420 425 430

Leu Glu Lys Thr Lys Tyr Pro Asn Ser Asp Leu Asn Trp Lys Lys Phe

435 440 445

Asp Asp Lys Tyr His Phe Ser Ser Gln Phe Thr Ala Asp Leu Phe Ala

450 455 460

Met Asn His Thr Asp Phe Ile Ile Thr Ser Thr Phe Gln Glu Ile Ala

465 470 475 480

Gly Ser Lys Asn Thr Val Gly Gln Tyr Glu Ser His Thr Ala Phe Thr

485 490 495

Met Pro Gly Leu Tyr Arg Val Val His Gly Ile Asp Ser Phe Asp Pro

500 505 510

Lys Phe Asn Ile Val Ser Pro Gly Ala Asp Met Ser Ile Tyr Phe Pro

515 520 525

Tyr Thr Glu Lys Glu Lys Arg Leu Thr Asn Phe His Pro Glu Ile Glu

530 535 540

Glu Leu Leu Tyr Ser Pro Val Glu Asn Lys Asp His Leu Cys Val Leu

545 550 555 560

Lys Asp Arg Asn Lys Pro Ile Leu Phe Thr Met Ala Arg Leu Asp Arg

565 570 575

Val Lys Asn Leu Thr Gly Leu Val Glu Trp Tyr Ala Lys Asn Ala Arg

580 585 590

Leu Arg Glu Leu Val Asn Leu Val Val Val Gly Gly Asp Arg Arg Lys

595 600 605

Glu Ser Lys Asp Leu Glu Glu Gln Ala Glu Met Lys Lys Met Tyr Asp

610 615 620

Leu Ile Glu Thr Tyr Asn Leu Asn Gly Gln Phe Arg Trp Ile Ser Ser

625 630 635 640

Gln Met Asn Arg Val Arg Asn Gly Glu Leu Tyr Arg Tyr Ile Ala Asp

645 650 655

Thr Arg Gly Ala Phe Val Gln Pro Ala Phe Tyr Glu Ala Phe Gly Leu

660 665 670

Thr Val Val Glu Ser Met Thr Cys Gly Leu Pro Thr Phe Ala Thr Cys

675 680 685

Asn Gly Gly Pro Phe Glu Ile Ile Val Asn Gly Lys Ser Gly Phe His

690 695 700

Ile Asp Pro Asn Gln Gly Asp Lys Ala Ala Asp Met Leu Val Asn Phe

705 710 715 720

Phe Glu Lys Ser Lys Glu Asp Pro Ser Tyr Trp Asp Ala Ile Ser Lys

725 730 735

Gly Gly Leu Gln Arg Ile Leu Glu Lys Tyr Thr Trp Gln Ile Tyr Ser

740 745 750

Gln Lys Val Ile Thr Leu Ser Gly Ile Tyr Gly Phe Trp Lys Tyr Ala

755 760 765

Thr Lys Asn Asp Lys Val Ala Ser Ala Lys Lys Arg Tyr Leu Glu Met

770 775 780

Phe Tyr Glu Leu Gly Phe Lys Lys Ser Ala Glu Lys Val Pro Leu Ala

785 790 795 800

Ile Asp Glu

<210> 3

<211> 3926

<212> DNA

<213> Nicotiana tabacum

<400> 3

atggcaggca gtggtcttag cattaaggaa agtttgggagg aatccatttt ggctcatcca 60

gatgaaattt tggctctcaa gtcaaggtac attactgcat aatgatatta agacctagaa 120

gcggatccaa gattttgtta catttttgaa attataagtt tagaatctaa tatttgttat 180

cgcttgtttc cttattatct tgctgttgtt actgcctgtt gctactagtt tctgttcatc 240

cttccttgag ctgagtttct atcggaaaca acctctctac tctcaaagta ggaataagtt 300

atgcgtacac actaccctcc ccagactcca cttgtgtaat ttactgagtt tgttgttgtt 360

gttgttgtaa tctaatactt gttagaattt tactgatttt tcacatatat atctatgacc 420

catgtcgaaa atactatagc tcatgtgcta aatacattag taccattgtt ttgtaattgt 480

tttggttttg gaacaggatt gaaactgaag ggaaaggggt aatgaaacca gttgatctct 540

tgaaccattt ggtttctgtt actagtaaaa caaatggagt aaatgttgta cctagtgcac 600

ttgtggaagt tctcagttgc agccaagaag ctgtgattgt accaccaaaa ctagcactag 660

ctgtacgtcc gaggcccggt gtatgggagt acttgtcact gaatcttaag acaaagaaag 720

tggctgaatt gagcattcct gagtaccttc aattgaaaga gaatactgtt gatgaaaggt 780

840 aaagtaatag tctgcgattt cgctttgtga

tttgtgtatc aattatgtta ccagtggaaa catcttggag ttggattttg agccatttac 900

aactgttaca acaccaaaaa cactttctga ctctattggc aatggtttgg agtttcttaa 960

tcgccacatt gcttcgaaaa tgtttcttga taaggagatt gccaagtgcc tccttgactt 1020

1080

1140

aagcattcaa agcctggaga gtttccaact tgttctgaaa aaagcagagg aatatttgca 1200

cacactgaat ccagaaactc catactccaa atttgaatcc aagtttgaag agattggctt 1260

ggaaagaggg tggggaaaca ccgctgaacg cgtgcaagac accattagtc atcttttgca 1320

tctccttgag gctcctaacg cgtcttcctt ggaaaatttc cttggtagaa tcccattggt 1380

tttcaatgtt gtgattctca ccccacatgg ttatttttgct caagataatg tcttgggcta 1440

tcctgacact ggtggccagg tttgtgtccg atataacata tcaagaaatt ttgcattctt 1500

1560

ggttgtttac attcttgatc aagttccagc tatggagcgt gagatgcttc atcgtatgaa 1620

gcttcaagga ctcgacgata tcatccctcg catccttgtt gtaagtgccc ttaattttcc 1680

1740

ctttttgttg ttttatatca ggtaactagg ctgctgcctg atgctgtagg aaccacttgt 1800

ggcgagtgga tggagaaagt atatggggca gaacattctc atataattcg tgttccattt 1860

agaactgaga aaggaatgtt gcgcaaatgg atctcacgat tcgaagtctg gccatacatg 1920

gaaactttca ctgaggttgg aacataaaaa caaataaaaa tcattggaat gttcttctgc 1980

atttgaaaat gtcttgctaa ctaaagactc atttttaaat taatcatcag gatgttgcag 2040

aagaacttgt caaagaattg caagctaaac cagacttgat aattggaaac tacagtgagg 2100

gaaatcttgc tgcctcattg cttgctaaga aatttggggc tactcagtgt actattgctc 2160

atgccttgga aaaaactaag tatccaaact ctgaccttaa ttggaagaag tttgatgaca 2220

agtatcattt ctcaagtcag ttcactgctg atctttttgc catgaatcac actgatttca 2280

ttatcaccag cactttccaa gaaattgctg gaaggtaaaa gcaaatgcac accatcatag 2340

tatttcatat ttttacccta gtttatacta tttccatttg tcaactccaa cttgtttggg 2400

attgaaccat agttgttgtt tgtttatact atttccattc gccgacccca acttatttgg 2460

gactgagaca taattgttgt tattattgtt tgtttgttta tactatttcc attctcagac 2520

2580

tgctatttcc gttcaccgac cccaacttat ttgggactga ggtgtagaag tagtcgttgt 2640

tgtttgttta tacgacttcc aattgatatt cgaatgtttt tatttttgca gcaagaacac 2700

tgtaggacag tatgagagtc atactgcttt taccatgcct ggattgtatc gagtagtcca 2760

tggaatcaat tcgtttgatc caaagttcaa cattgtctcc cctggggctg atatgtcaat 2820

ctacttccct tacactgaga aggagaaaag actaaccaac ttccacccgg aaattgaaga 2880

actcctctac agtcctgttg agaataagga ccacttgtta gtcttcttta tttcattcat 2940

ttttctacac cttttttttc aacagattga ttgattggtt cttatcaacg taaacagatg 3000

tgtgttgaag gaccagaaca agccaattct ctttaccatg gcaaggctag atcgcgtgaa 3060

gaatctaaca gggctcgtgg aatggtatgc aaagaatgca aggctaaggg agctcgttaa 3120

ccttgtggtt gtaggcggag acagaaggaa agaatccaaa gatttagaag agcaagcaga 3180

gatgaagaag atgtatgatc ttatcgaaac atacaacctg aatggccaat tcaggtggat 3240

ttcttcccaa atgaatcgtg tgaggaacgg agaactttat cgatacattg cagacacgag 3300

gggtgctttc gttcaaccag cattttatga ggcatttggt ttgacagttg ttgagtctat 3360

3420

tggaaaatct ggtttccata ttgatcctaa tcaaggtgac aaggctgctg atatgttggt 3480

taatttcttc gaaaaatcta aagaagatcc aagttattgg gatactattt ccaagggtgg 3540

tctgcagcgt attcttgaaa agtaagcttt tgcatttgat tagcacaagt gtacaaccaa 3600

gatttaacttatgaacaaac taaaactaac

3720

3780

atttgggttt aagaaatcag taagtgtcac ttctgtattt tgtttgagct tgtttgtaaa 3840

gtttggcaat cttctgctaa tttgtactat atttgttgac ttgtgcattt caggctgaga 3900

aagttccatt ggctattgat gaatag 3926

<210> 4

<211> 803

<212> PROTEIN

<213> Nicotiana tabacum

<400> 4

Met Ala Gly Ser Gly Leu Ser Ile Lys Glu Ser Leu Glu Glu Ser Ile

1 5 10 15

Leu Ala His Pro Asp Glu Ile Leu Ala Leu Lys Ser Arg Ile Glu Thr

20 25 30

Glu Gly Lys Gly Val Met Lys Pro Val Asp Leu Leu Asn His Leu Val

35 40 45

Ser Val Thr Ser Lys Thr Asn Gly Val Asn Val Val Pro Ser Ala Leu

50 55 60

Val Glu Val Leu Ser Cys Ser Gln Glu Ala Val Ile Val Pro Pro Lys

65 70 75 80

Leu Ala Leu Ala Val Arg Pro Arg Pro Gly Val Trp Glu Tyr Leu Ser

85 90 95

Leu Asn Leu Lys Thr Lys Lys Val Ala Glu Leu Ser Ile Pro Glu Tyr

100 105 110

Leu Gln Leu Lys Glu Asn Thr Val Asp Glu Ser Gly Asn Ile Leu Glu

115 120 125

Leu Asp Phe Glu Pro Phe Thr Thr Val Thr Thr Pro Lys Thr Leu Ser

130 135 140

Asp Ser Ile Gly Asn Gly Leu Glu Phe Leu Asn Arg His Ile Ala Ser

145 150 155 160

Lys Met Phe Leu Asp Lys Glu Ile Ala Lys Cys Leu Leu Asp Phe Leu

165 170 175

Arg Asn His Asn Tyr Lys Gly Lys Ser Leu Met Val Lys Glu Ser Ile

180 185 190

Gln Ser Leu Glu Ser Phe Gln Leu Val Leu Lys Lys Ala Glu Glu Tyr

195 200 205

Leu His Thr Leu Asn Pro Glu Thr Pro Tyr Ser Lys Phe Glu Ser Lys

210 215 220

Phe Glu Glu Ile Gly Leu Glu Arg Gly Trp Gly Asn Thr Ala Glu Arg

225 230 235 240

Val Gln Asp Thr Ile Ser His Leu Leu His Leu Leu Glu Ala Pro Asn

245 250 255

Ala Ser Ser Leu Glu Asn Phe Leu Gly Arg Ile Pro Leu Val Phe Asn

260 265 270

Val Val Ile Leu Thr Pro His Gly Tyr Phe Ala Gln Asp Asn Val Leu

275 280 285

Gly Tyr Pro Asp Thr Gly Gly Gln Val Val Tyr Ile Leu Asp Gln Val

290 295 300

Pro Ala Met Glu Arg Glu Met Leu His Arg Met Lys Leu Gln Gly Leu

305 310 315 320

Asp Asp Ile Ile Pro Arg Ile Leu Val Val Thr Arg Leu Leu Pro Asp

325 330 335

Ala Val Gly Thr Thr Cys Gly Glu Trp Met Glu Lys Val Tyr Gly Ala

340 345 350

Glu His Ser His Ile Ile Arg Val Pro Phe Arg Thr Glu Lys Gly Met

355 360 365

Leu Arg Lys Trp Ile Ser Arg Phe Glu Val Trp Pro Tyr Met Glu Thr

370 375 380

Phe Thr Glu Asp Val Ala Glu Glu Leu Val Lys Glu Leu Gln Ala Lys

385 390 395 400

Pro Asp Leu Ile Ile Gly Asn Tyr Ser Glu Gly Asn Leu Ala Ala Ser

405 410 415

Leu Leu Ala Lys Lys Phe Gly Ala Thr Gln Cys Thr Ile Ala His Ala

420 425 430

Leu Glu Lys Thr Lys Tyr Pro Asn Ser Asp Leu Asn Trp Lys Lys Phe

435 440 445

Asp Asp Lys Tyr His Phe Ser Ser Gln Phe Thr Ala Asp Leu Phe Ala

450 455 460

Met Asn His Thr Asp Phe Ile Ile Thr Ser Thr Phe Gln Glu Ile Ala

465 470 475 480

Gly Ser Lys Asn Thr Val Gly Gln Tyr Glu Ser His Thr Ala Phe Thr

485 490 495

Met Pro Gly Leu Tyr Arg Val Val His Gly Ile Asn Ser Phe Asp Pro

500 505 510

Lys Phe Asn Ile Val Ser Pro Gly Ala Asp Met Ser Ile Tyr Phe Pro

515 520 525

Tyr Thr Glu Lys Glu Lys Arg Leu Thr Asn Phe His Pro Glu Ile Glu

530 535 540

Glu Leu Leu Tyr Ser Pro Val Glu Asn Lys Asp His Leu Cys Val Leu

545 550 555 560

Lys Asp Gln Asn Lys Pro Ile Leu Phe Thr Met Ala Arg Leu Asp Arg

565 570 575

Val Lys Asn Leu Thr Gly Leu Val Glu Trp Tyr Ala Lys Asn Ala Arg

580 585 590

Leu Arg Glu Leu Val Asn Leu Val Val Val Gly Gly Asp Arg Arg Lys

595 600 605

Glu Ser Lys Asp Leu Glu Glu Gln Ala Glu Met Lys Lys Met Tyr Asp

610 615 620

Leu Ile Glu Thr Tyr Asn Leu Asn Gly Gln Phe Arg Trp Ile Ser Ser

625 630 635 640

Gln Met Asn Arg Val Arg Asn Gly Glu Leu Tyr Arg Tyr Ile Ala Asp

645 650 655

Thr Arg Gly Ala Phe Val Gln Pro Ala Phe Tyr Glu Ala Phe Gly Leu

660 665 670

Thr Val Val Glu Ser Met Thr Cys Gly Leu Pro Thr Phe Ala Thr Cys

675 680 685

Asn Gly Gly Pro Phe Glu Ile Ile Val Asn Gly Lys Ser Gly Phe His

690 695 700

Ile Asp Pro Asn Gln Gly Asp Lys Ala Ala Asp Met Leu Val Asn Phe

705 710 715 720

Phe Glu Lys Ser Lys Glu Asp Pro Ser Tyr Trp Asp Thr Ile Ser Lys

725 730 735

Gly Gly Leu Gln Arg Ile Leu Glu Lys Tyr Thr Trp Gln Ile Tyr Ser

740 745 750

Gln Lys Val Ile Thr Leu Ser Gly Ile Tyr Gly Phe Trp Lys Tyr Ala

755 760 765

Thr Lys Asn Asp Lys Val Ala Ser Ala Lys Lys Arg Tyr Leu Glu Met

770 775 780

Phe Tyr Glu Phe Gly Phe Lys Lys Ser Ala Glu Lys Val Pro Leu Ala

785 790 795 800

Ile Asp Glu

<210> 5

<211> 4532

<212> DNA

<213> Nicotiana tabacum

<400> 5

atggctgaac gtgctctgac tcgtgttcac agccttcgtg aacgtcttga tgccactttg 60

gctgcacatc gcaatgagat attgctgttt ctttcaaggt attgcctaag tagtgttctt 120

gtttcctaca aaagattcag ttggtgttca aaaaacgata tgtgatttga tttatctgcc 180

taagtcttgg tagtcataat tatccggtac ctgtgctggt gcgagttagc tggttcggaa 240

actactctta tgaaaacgag agatttagtt ggtgttgtct gcaattctgt agtatggact 300

attaagcaga tagatcatgt ttgatatcga aaaggaatgt atatgtgatg ttacttgaac 360

tggttttgggt tattacagga ttgaaagcca tggaaaaggg atcttgaaac ctcaccagct 420

attggctgag ttcgatgcaa ttcgccaaga tgacaaaaag aagctgaatg atcatgcatt 480

tgaagaactc ctgaaatcta ctcaggtaat tttgattttg gctaaatgtg ttaccaagct 540

gaatgatcat gcatttgagt ttgtgtccga ctactacaat gatatgttat accaggaagc 600

660

tgtccgtgtg aatgttaatg ctctagtcgt tgaggagctg accgtccctg agtatttgca 720

ttttaaggaa gaacttgttg atggaacgta agttttagtc tcttatttga tactatgtta 780

gagaataggc agtggattca atttatcagt gttgtttttt acctaatgca gctccaatgg 840

aaatttcgtt ctcgagttgg attttgagcc cttcactgca tcctttccta aaccgaccct 900

caccaaatct attgggaatg gagttgaatt cctcaatagg cacctttctg cgaaaatgtt 960

ccatgacaag gaaagcatga ccccgcttct tgaatttctt cgggttcaca attataaggg 1020

caaggtaact ttgttattcc cattcatata tatgttcagt ttgtgcttat catgcgccca 1080

1140

gaatgacaga atacagaatt taaccactct gcaaaatgtc ctaaggaagg cagaggaata 1200

ccttattatg cttccccctg aaactccatt ttccgaattc gaacacaagt tccaagaaat 1260

tggattggag aagggatggg gcgacactgc ggagcgcgtg ctagagatga tatgcatgct 1320

tcttgatcta cttgaggctc ccgactcctg tactcttgag aagttcctag ggagaattcc 1380

tatggtgttc aacgtggtta tccttttcccc ccatggatat ttcgcccagg aaaatgtctt 1440

gggttatccc gacactggtg gccaggtgca ttactttagt ctttgtccgt gagtctatgt 1500

tgctcagatc ctctacaatg ccactgtacc cgtgtaggat actccaaata taatgcattt 1560

ttggaggatc tgtcaccggt gcaatggcat tttggaggtc ggagcaacaa acaactgcta 1620

gtatgcttct aaagcttgct tccataaatg ctaaggtcct tcacccgtaa tgtgcaggtt 1680

gtctacatat tagatcaagt tccagccttg gagcgtgaaa tgcttaaacg cctaaaggag 1740

caaggacttg atataacacc gcgtattctt attgttagta tttcttgtac ttgtaattgc 1800

tgcggattac acaaaatttt ctctttattg gcaacttatc ttgatattat tcccaggtta 1860

ctcgtctgct gcctgatgca gttggaacaa cttgtggtca gcggcttgag aaggtgtatg 1920

gagccgagca ctcacatatt cttagggtcc cctttaggac cgagaagggc attgttcgca 1980

aatggatatc tcgctttgaa gtgtggccat acatggagac tttcactgag gtgacactaa 2040

gcttccttgt atttgtctat cttctaattg gtattaggaa caatttgcta attattaacg 2100

ctttggcttt tcgtacatca ggatgttgca aaagaacttg ctgcagaact gcaggccaag 2160

ccagatttga taattggcaa ctatagcgag ggaaatcttg tggcttcatt gctggctcac 2220

aagttaggcg taacgcaggt ctgtgttatt tttcacctct tataaatctg attgtatttc 2280

cattagtctg gaactaaaag tactaaaatt ttcttttctt cgctgtgtta tttgccttct 2340

gcagtgcacc attgcccatg cattggagaa aacaaagtat cctgattctg acatctactg 2400

gaaaaaattt gacgaaaaat accatttctc gtcccagttt accgctgatc ttattgcaat 2460

2520

caattgctaa ttcggttgca gtaacatttt gttcgatttc ttccccttat gcttaaccta 2580

ataccctaat gaattttcca gcaaggacac tgtcggacag tacgagagtc accaggcatt 2640

cacaatgcct ggattgtaca gagtcgttca cggcattgat gtgttcgatc ccaaattcaa 2700

cattgtctca cctggagctg atataaacct gtatttccca tattccgaga aggaaaagag 2760

attgacagca cttcacccag aaattgagga gcttctgtac agtgatgttg agaacgagga 2820

acatctgtaa gtttctaact tactcgtacc gtcagtggca gagccagaat tttcattaaa 2880

atggggtcaa aatataaaga cataaattca caaagaagcc aaggggtgtc aatatgtagt 2940

ataaatatat taaaaaaatt acctagctac acaatgtaat ttttccgacaa aggggtatcg 3000

gttgcacttc ttgaatacat gtggctctgc cactgggtac agttacaaag tcctgttacc 3060

3120

gaataagcca atcttattca caatggcgag attggatcgt gtgaagaact taaccggact 3180

tgttgagtgg tacgccaaga acgcacggct aagggagttg gttaaccttg ttgtcgttgg 3240

3300

tgagctaata aagactcaca acttaaatgg ccaattcaga tggatttctt cacagatgaa 3360

ccgagtaagg aacggcgaac tctaccgata cattgccgac actaggggag ctttcgtgca 3420

gcctgcattc tatgaggctt tcggtttgac tgttgttgag gccatgacct gtggtttgcc 3480

tacatttgca actaatcatg gcggtccagc tgagatcatc gttaacggaa aatccggctt 3540

ccatatcgat ccatatcacg gtgagcaagc tgctgatctg ctagctgatt tctttgagaa 3600

3660

agagaagtaa gcaactcttt cttgactcta gtcattcaaa ttaacttggg atttgaggca 3720

tagttgattg ataatttatc gcgtctctac tactatatac aggtacacgt ggcaaatcta 3780

3840

tgatcgtcta gaaatccgtc gatatctaga aatgttttat gctctcaaat accggaagat 3900

ggtgagttct tctgcttcct gctcttctca tagtgtttaa tatacacttg attgattgca 3960

ttcacttaga ctaagttgct cggacacggg tgtggatgtc cgacacgagt gcggatctag 4020

4080

cgggtgcgag aatccggcta aaaataattt taaaaaaaat tatctctaaa ttatgagata 4140

ttatgtggaa tacttacgta taacttgtaa agtgtagatt ttttttaatt ctcaagttgt 4200

agattagtaa atgattgatt tcctagataa gtatgctatt ttcttcaaat ttactcttct 4260

gatttcgaaa atcaaattgt atctcgtctc gaatttttcc gtccgttatg gtcaaagtac 4320

ccaaaatcgt ttgaccaaat cggtacggat cccataccca cacccacact agtgtcgtat 4380

tgacacgggt gccgcaccta aactgctatg tcggagcaac ttagcactta gagaatcatt 4440

gatgttaaat ttttcttaatt cttgaatctg ctaatgaaga ttttatcttg gtttttgttt 4500

aggctgaagc tgttccattg gctgctgaat ga 4532

<210> 6

<211> 805

<212> PROTEIN

<213> Nicotiana tabacum

<400> 6

Met Ala Glu Arg Ala Leu Thr Arg Val His Ser Leu Arg Glu Arg Leu

1 5 10 15

Asp Ala Thr Leu Ala Ala His Arg Asn Glu Ile Leu Leu Phe Leu Ser

20 25 30

Arg Ile Glu Ser His Gly Lys Gly Ile Leu Lys Pro His Gln Leu Leu

35 40 45

Ala Glu Phe Asp Ala Ile Arg Gln Asp Asp Lys Lys Lys Leu Asn Asp

50 55 60

His Ala Phe Glu Glu Leu Leu Lys Ser Thr Gln Glu Ala Ile Val Leu

65 70 75 80

Pro Pro Trp Val Ala Leu Ala Ile Arg Leu Arg Pro Gly Val Trp Glu

85 90 95

Tyr Val Arg Val Asn Val Asn Ala Leu Val Val Glu Glu Leu Thr Val

100 105 110

Pro Glu Tyr Leu His Phe Lys Glu Glu Leu Val Asp Gly Thr Ser Asn

115 120 125

Gly Asn Phe Val Leu Glu Leu Asp Phe Glu Pro Phe Thr Ala Ser Phe

130 135 140

Pro Lys Pro Thr Leu Thr Lys Ser Ile Gly Asn Gly Val Glu Phe Leu

145 150 155 160

Asn Arg His Leu Ser Ala Lys Met Phe His Asp Lys Glu Ser Met Thr

165 170 175

Pro Leu Leu Glu Phe Leu Arg Val His Asn Tyr Lys Gly Lys Thr Met

180 185 190

Met Leu Asn Asp Arg Ile Gln Asn Leu Thr Thr Leu Gln Asn Val Leu

195 200 205

Arg Lys Ala Glu Glu Tyr Leu Ile Met Leu Pro Pro Glu Thr Pro Phe

210 215 220

Ser Glu Phe Glu His Lys Phe Gln Glu Ile Gly Leu Glu Lys Gly Trp

225 230 235 240

Gly Asp Thr Ala Glu Arg Val Leu Glu Met Ile Cys Met Leu Leu Asp

245 250 255

Leu Leu Glu Ala Pro Asp Ser Cys Thr Leu Glu Lys Phe Leu Gly Arg

260 265 270

Ile Pro Met Val Phe Asn Val Val Ile Leu Ser Pro His Gly Tyr Phe

275 280 285

Ala Gln Glu Asn Val Leu Gly Tyr Pro Asp Thr Gly Gly Gln Val Val

290 295 300

Tyr Ile Leu Asp Gln Val Pro Ala Leu Glu Arg Glu Met Leu Lys Arg

305 310 315 320

Leu Lys Glu Gln Gly Leu Asp Ile Thr Pro Arg Ile Leu Ile Val Thr

325 330 335

Arg Leu Leu Pro Asp Ala Val Gly Thr Thr Cys Gly Gln Arg Leu Glu

340 345 350

Lys Val Tyr Gly Ala Glu His Ser His Ile Leu Arg Val Pro Phe Arg

355 360 365

Thr Glu Lys Gly Ile Val Arg Lys Trp Ile Ser Arg Phe Glu Val Trp

370 375 380

Pro Tyr Met Glu Thr Phe Thr Glu Asp Val Ala Lys Glu Leu Ala Ala

385 390 395 400

Glu Leu Gln Ala Lys Pro Asp Leu Ile Ile Gly Asn Tyr Ser Glu Gly

405 410 415

Asn Leu Val Ala Ser Leu Leu Ala His Lys Leu Gly Val Thr Gln Cys

420 425 430

Thr Ile Ala His Ala Leu Glu Lys Thr Lys Tyr Pro Asp Ser Asp Ile

435 440 445

Tyr Trp Lys Lys Phe Asp Glu Lys Tyr His Phe Ser Ser Gln Phe Thr

450 455 460

Ala Asp Leu Ile Ala Met Asn His Thr Asp Phe Ile Ile Thr Ser Thr

465 470 475 480

Phe Gln Glu Ile Ala Gly Ser Lys Asp Thr Val Gly Gln Tyr Glu Ser

485 490 495

His Gln Ala Phe Thr Met Pro Gly Leu Tyr Arg Val Val His Gly Ile

500 505 510

Asp Val Phe Asp Pro Lys Phe Asn Ile Val Ser Pro Gly Ala Asp Ile

515 520 525

Asn Leu Tyr Phe Pro Tyr Ser Glu Lys Glu Lys Arg Leu Thr Ala Leu

530 535 540

His Pro Glu Ile Glu Glu Leu Leu Tyr Ser Asp Val Glu Asn Glu Glu

545 550 555 560

His Leu Cys Val Leu Lys Asp Arg Asn Lys Pro Ile Leu Phe Thr Met

565 570 575

Ala Arg Leu Asp Arg Val Lys Asn Leu Thr Gly Leu Val Glu Trp Tyr

580 585 590

Ala Lys Asn Ala Arg Leu Arg Glu Leu Val Asn Leu Val Val Val Gly

595 600 605

Gly Asp Arg Arg Lys Glu Ser Lys Asp Leu Glu Glu Gln Ala Glu Met

610 615 620

Lys Lys Met Tyr Glu Leu Ile Lys Thr His Asn Leu Asn Gly Gln Phe

625 630 635 640

Arg Trp Ile Ser Ser Gln Met Asn Arg Val Arg Asn Gly Glu Leu Tyr

645 650 655

Arg Tyr Ile Ala Asp Thr Arg Gly Ala Phe Val Gln Pro Ala Phe Tyr

660 665 670

Glu Ala Phe Gly Leu Thr Val Val Glu Ala Met Thr Cys Gly Leu Pro

675 680 685

Thr Phe Ala Thr Asn His Gly Gly Pro Ala Glu Ile Ile Val Asn Gly

690 695 700

Lys Ser Gly Phe His Ile Asp Pro Tyr His Gly Glu Gln Ala Ala Asp

705 710 715 720

Leu Leu Ala Asp Phe Phe Glu Lys Cys Lys Thr Glu Pro Ser His Trp

725 730 735

Glu Thr Ile Ser Thr Gly Gly Leu Lys Arg Ile Gln Glu Lys Tyr Thr

740 745 750

Trp Gln Ile Tyr Ser Glu Arg Leu Leu Thr Leu Ala Ala Val Tyr Gly

755 760 765

Phe Trp Lys His Val Ser Lys Leu Asp Arg Leu Glu Ile Arg Arg Tyr

770 775 780

Leu Glu Met Phe Tyr Ala Leu Lys Tyr Arg Lys Met Ala Glu Ala Val

785 790 795 800

Pro Leu Ala Ala Glu

805

<210> 7

<211> 5901

<212> DNA

<213> Nicotiana tabacum

<400> 7

atgcttttta tgggagtaaa ttttatggcc ggtcattcaa ctttgtgttc attacgcaaa 60

120

180 atcattcaac tatgtgttta ttatctaaaa

aaatcatttt actttactct atttatcaca aaagtcacct tggccagatt ttataatagg 240

cttttatctt ttgttacaca aaaattattt tactttactc tatttatcac aaaagtcacc 300

ttggccagat tttataatag gcttttatct tttgttacac aaaaattatt ttactttact 360

ctatttatca caaaagtcac cttggccaga tttttacaata cttttacctt aaaagactat 420

tatgcccttg acattataaa tcctctcatt tatataatac cttctatatg atacactata 480

taatattattt ttacctaggt attttactta taattaaaat aatattaaat tattttattt 540

atctatttta taatatattc atacatttaa ttttttcatg gcaaatcact ttgtttaatc 600

660

tttgaaataa taacaaacag atttgtttaa caaatgatag ttttttttta tagtcaataa 720

aatttttaaa aaaattcaaa gatatttgtt tttaatatta atatttttaa agctttatct 780

840

ttatttattg gattaatgag tatggcttgg ctgataaaaa gctttgattt tataattttc 900

attaaaaata ttttattaag ctagtacctg acaaatttaa tatcttgaaa attaacgtta 960

agaaaaaatt aaatataaaa atatattata aaaataataa ataaataata tcaagttatt 1020

ttaattataa ataaaataca tggttaaaaa tatattatat agcatataat atagaaggta 1080

ttacataaat gagatgattt aaagggcata atagactttt caggtgaatg atttgtaaaa 1140

tatggttaaa gtgattattg tgataattag agcatagtaa aataattttt atgtaacaaa 1200

agaaaaaaaa aatgactttt gggtaatgaa cataaatttg aataactttt acgtaacaaa 1260

agaataaaat aaattttgga taataaacat aaaattgaat gaccacctat aaaatttatt 1320

atttttttgg gctcttcttg atttgatttt ttagtttagc ctttgcagta atcttggttg 1380

tcacgcgtag cgttgtgctt tcgccacata agtatttagt agacttaatt aatgtcatta 1440

tatcggttgg tgtggtttta attacttaac tgtactatta tattaggtgg aaggtttgaa 1500

1560

1620

gagaaacaat ctctctcacc tacacggtac gggtaaggta tgcgtatacg cttatcctcc 1680

ctacactcca tttgtgggac tattgttgtt attttggata agctgaggta tccatcttct 1740

actaactgca ctagtttatt ttttttgctg tttacagttg aaacaattgt ctgaggattt 1800

ctcacctgct gaatcaactg caatggctga acgtgtgctg actcgtgttc acagccttcg 1860

tgaacgtctt gatgctactt tggctgctca tcgcaatgag atattactgt ttctttcaag 1920

gtatagccaa agatagtatt cttgttaact aaaaaagatt cagttggtgt tcaaaaaacg 1980

atacgtttat ctgcctaagt cttggtagtc agaattatcc ggtacctatg ctggtgtgag 2040

ttagctggct aggaaaccac tcttatgaaa acaagagatt tagttagagt tgtctgtaat 2100

2160

aaagccatgg aaaagggatc ttgaaaccgc atcagctatt ggctgagttt gatgcaattc 2220

gccaagatga caaaaagaaa ctgaatgatc atgcatttga agaactcctg aagtccactc 2280

2340

tgagcttgtg tctgaatacg acgatgatat gttatactag gaagcaattg ttctgccacc 2400

ttgggttgca cttgcgattc gtttgaggcc tggtgtgtgg gaatatgtcc gtgtgaatgt 2460

caatgcgcta gtcgttgagg agctgactgt ccctgagtat ttgcatttca aggaagaact 2520

2580

2640

agttggattt tgagcccttc accgcatcct ttcctaaacc aaccctcacc aaatctatcg 2700

gaaatggagt tgaattcctc aataggcacc tctctgcgaa aatgttccat gacaaggaaa 2760

gcatgacccc gcttcttgaa tttcttcggg ttcacaatta taagggcaag gtgacttgct 2820

atttccattt atctataggt tcggtttgtg cttatcatgc gcccaatgac atatgaatat 2880

gcgctaaagg atagatatat gatttccttt gcagacaatg atgctgaacg acagaataca 2940

gaatttaacc acactgcaaa atgtcctaag gaaggcagag gaatacctca ttatgcttcc 3000

ccctgaaact ccattttccg aattcgaaca caagttccaa gaaattggat tggagaaggg 3060

atggggcgac actgcagagc gcgtgctgga gatgatatgc atgcttcttg atctcctcga 3120

3180

ggttatcctt tccccccg gatatttcgc cggaaaat gtcttgggtt atcccgacac 3240

tggtggccag gtgcattact ttaatcttta tccgtgagtc tatgtttgtt cgaatcctct 3300

agaaatgtca ctgtacctat gtaggatact ccaaatataa tgcattttgg ggggatctgt 3360

tatgggtgcg atggcatttt tggaggtcgg agcaacaaac aattgctatg tattcttcta 3420

3480

agatcaagtt ccagccttgg agcgtgaaat gcttaagcgc ctaaaggagc aaggacttga 3540

ttgttagtat ttcctgtact tgtaattact gcggattaca 3600

caaaatttcc tttttatctt cttaacaact tatcttgatg gtattcccag gttactcgtc 3660

tgctacctga tgcagttgga acgacttgtg gtcagcggct tgagaaggtg tatggagccg 3720

agcactcaca tattctgagg gtccccttta ggactgagaa gggcattgtt cgtaaatgga 3780

tctctcgctt tgaagtgtgg ccatatatgg agactttcac tgaggtgaca ctaaaacttc 3840

cttatatttg tctatcttct aattggtatt aggaataatt tgttaattgt taactctttg 3900

tcttttcgta catcaggatg tcgcaaaaga acttgctgca gaattgcagg ccaagccaga 3960

tttgataata ggcaactata gcgagggaaa tcttgtggct tcattgctcg ctcataagtt 4020

aggcgtaaca caggtctgtg ttgtttttca ctctcttaaa gatctgattg catttccatt 4080

agtctggaac tagaagtact aaaaagttct tttcttcact gtgttatttg ccgtcggcag 4140

tgcaccatag ctcatgcatt ggagaaaaca aagtatcctg attctgacat ctactggaaa 4200

aaattcgatg aaaaatacca tttctcgtcc cagtttaccg ctgatcttat tgcaatgaat 4260

4320

ttgctacttc gactgcaaca gcattgtgtt cccatttctt tcccttatgc ttaacctaat 4380

accgtcatga attttccagc aaggacactg tcggacagta cgagagtcat caggcattca 4440

caatgcccgg attgtacaga gttgttcacg gcattgatgt gttcgacccc aaattcaaca 4500

ttgtctcacc tggagctgac ataaacctct atttcccata ttccgagaag gaaaagagac 4560

tgacagcact tcaccctgaa atcgaggagc tgctgtacag tgacattgag aacgaggaac 4620

atctgtaagt ttctacctta ctcgtacagt cagtggcgga gccagaattt tcactaaaat 4680

aaggtcaaaa tataaagaca taaatccaca aagaagccaa gggtgtcaat atatagtata 4740

aatacattaa aaaaattacc tatctacaca gtgtaatttt ccgacaaagg ggtgtcggtt 4800

gacactcctt gaatacatgt ggctctgcca ctgggtacag ttacaaagtt ctgttaccta 4860

tgtagatgag cttgtgctga acatgttgtg attttggcag gtgtgtgcta aaggacagga 4920

ataagccaat cttattcaca atggcgagat tggatcgtgt gaagaattta accggacttg 4980

ttgagtggta tgccaagaac gcacggctaa gggagttggt taaccttgtt gtggttggtg 5040

gagatcgaag gaaagaatcc aaagatttgg aagagcaaac agaaatgaaa aagatgtatg 5100

agctaataaa gactcacaat ttaaatggcc aattcagatg gatttcttca cagatgaacc 5160

gagtgaggaa cggtgaactc taccgataca ttgctgacac tagagggagct ttcgtgcagc 5220

ctgcattcta cgaggctttc ggtttgactg ttgttgaggc catgacctgt ggtttgccta 5280

catttgcaac taatcatggc ggtccagctg agatcatcgt taacggaaaa tctggcttcc 5340

acatcgatcc atatcacggt gagcaagctg ctgatctgct agctgatttc tttgagaaat 5400

gtaagacaga accttctcat tgggaaacca tttcaacggg tggcctgaag cgcatccaag 5460

agaagtaagc aactctttct tgactctagt cattgaaatt aactttcttg actctagtca 5520

5580

gatatataca ggtacacgtg gcaaatctac tcggagaggc tattgacatt ggctgctgtt 5640

tacgggttct ggaaacatgt ttctaagctt gatcgtctag aaatccgtcg atatcttgaa 5700

atgttttatg ctctcaaata ccgcaagatg gtgagttcct cttcttcctt gcccttctcc 5760

tagtgtttaa gatacaatat aattgattgc attatcttag agaatcatta atgttaaatt 5820

ttcttaattc ttgaatctgt taatgaagtt ttttctcttgg tttttgttta ggctgaagct 5880

gttccattgg ctgctgagtg a 5901

<210> 8

<211> 825

<212> PROTEIN

<213> Nicotiana tabacum

<400> 8

Met Leu Phe Met Gly Leu Lys Gln Leu Ser Glu Asp Phe Ser Pro Ala

1 5 10 15

Glu Ser Thr Ala Met Ala Glu Arg Val Leu Thr Arg Val His Ser Leu

20 25 30

Arg Glu Arg Leu Asp Ala Thr Leu Ala Ala His Arg Asn Glu Ile Leu

35 40 45

Leu Phe Leu Ser Arg Ile Glu Ser His Gly Lys Gly Ile Leu Lys Pro

50 55 60

His Gln Leu Leu Ala Glu Phe Asp Ala Ile Arg Gln Asp Asp Lys Lys

65 70 75 80

Lys Leu Asn Asp His Ala Phe Glu Glu Leu Leu Lys Ser Thr Gln Glu

85 90 95

Ala Ile Val Leu Pro Pro Trp Val Ala Leu Ala Ile Arg Leu Arg Pro

100 105 110

Gly Val Trp Glu Tyr Val Arg Val Asn Val Asn Ala Leu Val Val Glu

115 120 125

Glu Leu Thr Val Pro Glu Tyr Leu His Phe Lys Glu Glu Leu Val Asp

130 135 140

Gly Thr Ser Asn Gly Asn Phe Val Leu Glu Leu Asp Phe Glu Pro Phe

145 150 155 160

Thr Ala Ser Phe Pro Lys Pro Thr Leu Thr Lys Ser Ile Gly Asn Gly

165 170 175

Val Glu Phe Leu Asn Arg His Leu Ser Ala Lys Met Phe His Asp Lys

180 185 190

Glu Ser Met Thr Pro Leu Leu Glu Phe Leu Arg Val His Asn Tyr Lys

195 200 205

Gly Lys Thr Met Met Leu Asn Asp Arg Ile Gln Asn Leu Thr Thr Leu

210 215 220

Gln Asn Val Leu Arg Lys Ala Glu Glu Tyr Leu Ile Met Leu Pro Pro

225 230 235 240

Glu Thr Pro Phe Ser Glu Phe Glu His Lys Phe Gln Glu Ile Gly Leu

245 250 255

Glu Lys Gly Trp Gly Asp Thr Ala Glu Arg Val Leu Glu Met Ile Cys

260 265 270

Met Leu Leu Asp Leu Leu Glu Ala Pro Asp Ser Cys Thr Leu Glu Lys

275 280 285

Phe Leu Gly Arg Ile Pro Met Val Phe Asn Val Val Ile Leu Ser Pro

290 295 300

His Gly Tyr Phe Ala Gln Glu Asn Val Leu Gly Tyr Pro Asp Thr Gly

305 310 315 320

Gly Gln Val Val Tyr Ile Leu Asp Gln Val Pro Ala Leu Glu Arg Glu

325 330 335

Met Leu Lys Arg Leu Lys Glu Gln Gly Leu Asp Ile Thr Pro Arg Ile

340 345 350

Leu Ile Val Thr Arg Leu Leu Pro Asp Ala Val Gly Thr Thr Cys Gly

355 360 365

Gln Arg Leu Glu Lys Val Tyr Gly Ala Glu His Ser His Ile Leu Arg

370 375 380

Val Pro Phe Arg Thr Glu Lys Gly Ile Val Arg Lys Trp Ile Ser Arg

385 390 395 400

Phe Glu Val Trp Pro Tyr Met Glu Thr Phe Thr Glu Asp Val Ala Lys

405 410 415

Glu Leu Ala Ala Glu Leu Gln Ala Lys Pro Asp Leu Ile Ile Gly Asn

420 425 430

Tyr Ser Glu Gly Asn Leu Val Ala Ser Leu Leu Ala His Lys Leu Gly

435 440 445

Val Thr Gln Cys Thr Ile Ala His Ala Leu Glu Lys Thr Lys Tyr Pro

450 455 460

Asp Ser Asp Ile Tyr Trp Lys Lys Phe Asp Glu Lys Tyr His Phe Ser

465 470 475 480

Ser Gln Phe Thr Ala Asp Leu Ile Ala Met Asn His Thr Asp Phe Ile

485 490 495

Ile Thr Ser Thr Phe Gln Glu Ile Ala Gly Ser Lys Asp Thr Val Gly

500 505 510

Gln Tyr Glu Ser His Gln Ala Phe Thr Met Pro Gly Leu Tyr Arg Val

515 520 525

Val His Gly Ile Asp Val Phe Asp Pro Lys Phe Asn Ile Val Ser Pro

530 535 540

Gly Ala Asp Ile Asn Leu Tyr Phe Pro Tyr Ser Glu Lys Glu Lys Arg

545 550 555 560

Leu Thr Ala Leu His Pro Glu Ile Glu Glu Leu Leu Tyr Ser Asp Ile

565 570 575

Glu Asn Glu Glu His Leu Cys Val Leu Lys Asp Arg Asn Lys Pro Ile

580 585 590

Leu Phe Thr Met Ala Arg Leu Asp Arg Val Lys Asn Leu Thr Gly Leu

595 600 605

Val Glu Trp Tyr Ala Lys Asn Ala Arg Leu Arg Glu Leu Val Asn Leu

610 615 620

Val Val Val Gly Gly Asp Arg Arg Lys Glu Ser Lys Asp Leu Glu Glu

625 630 635 640

Gln Thr Glu Met Lys Lys Met Tyr Glu Leu Ile Lys Thr His Asn Leu

645 650 655

Asn Gly Gln Phe Arg Trp Ile Ser Ser Gln Met Asn Arg Val Arg Asn

660 665 670

Gly Glu Leu Tyr Arg Tyr Ile Ala Asp Thr Arg Gly Ala Phe Val Gln

675 680 685

Pro Ala Phe Tyr Glu Ala Phe Gly Leu Thr Val Val Glu Ala Met Thr

690 695 700

Cys Gly Leu Pro Thr Phe Ala Thr Asn His Gly Gly Pro Ala Glu Ile

705 710 715 720

Ile Val Asn Gly Lys Ser Gly Phe His Ile Asp Pro Tyr His Gly Glu

725 730 735

Gln Ala Ala Asp Leu Leu Ala Asp Phe Phe Glu Lys Cys Lys Thr Glu

740 745 750

Pro Ser His Trp Glu Thr Ile Ser Thr Gly Gly Leu Lys Arg Ile Gln

755 760 765

Glu Lys Tyr Thr Trp Gln Ile Tyr Ser Glu Arg Leu Leu Thr Leu Ala

770 775 780

Ala Val Tyr Gly Phe Trp Lys His Val Ser Lys Leu Asp Arg Leu Glu

785 790 795 800

Ile Arg Arg Tyr Leu Glu Met Phe Tyr Ala Leu Lys Tyr Arg Lys Met

805 810 815

Ala Glu Ala Val Pro Leu Ala Ala Glu

820 825

<210> 9

<211> 8323

<212> DNA

<213> Nicotiana tabacum

<400> 9

atggcgaatc caaagttcac aagagtacct agcatgaggg agagagttga ggatactctc 60

tctgctcacc gtaaccagct tgttgctctc ctctccaggt atattaataa actctatata 120

180

tcttgctttc gatgcatgat ttctgttgtg ttaaattgcg taaccatttt atctaaaagt 240

ttatgctgat aaacactttt aaattttaat atgtaaatta tattatgtct caacatcaac 300

atgtggatgg ccaaaaatat aaagcttaat tttcgttatt ttgaatgatt tttctctgcg 360

agtgttacgg tttgcgtaca cattacctaa acctcctccc tagtccccac ttgtgggaat 420

ttaatttttt ttttctttgt ttttttttgt tgttgttgtt gtctgagttc aattcctacc 480

atgttagctt ggcaaaaata agttggtaaa gcttgacccc aactagtttt agttgatcga 540

tttatttggt gatttatagt tcaataataa taattactat tagagaaagt tccagcagct 600

660

tttaagatac gtggcgcagg ggaaggggat attgcaacct caccacttga tcgatgagtt 720

caacaacgct gtatgtgatg acactgcttg tgagaagctc aaagatggtc cctttagtga 780

agtcttgaaa gctactcagg tatattcact aatccatggg aatcaagatg atactgtata 840

tctttattat ggtgtctttc agaaatttga cgatgatgaa atgcaacttt tctctgtttg 900

tcaccttatc cagactgtttt ttttattttt tatttttcat tttttaactt gaaatgctct 960

taatttcctt tgtttatcga taagaccggga tttacaatgt atgaacggag catcttaaga 1020

accttctgga atgaagatat aagatataaa acatggtgtc cgttttctcc tttgtggaat 1080

cagtgtacat atagactgtt attttggtcc cactttctgg atcttctgat cacaccttct 1140

catgcagagg cgagcttgat ggtttcaacc tttaaattct tactattgaa tccatttcac 1200

tttcgaaatt atgagttcga aatctaatat ttgttgaaat ttttgcaaat gttcacatat 1260

aagtttaagc tttgtgtcaa gaatactggg ctcaatggat tccaatagac caggctgtat 1320

ccgcctctgt ctccactctc cctgcatcca cttctttcgt gtgactaata atgcttaatg 1380

1440

caatctttaa cgggttatgc agtaccagca ttctgcggct gaaaaacagg aatctgagat 1500

ttacttgtct ctggctgaat ttcttgttca ttttgctaac aagtactttg gagttaatgc 1560

ttgctctctg ttgtcaaaat aggaagccat tgtgctgcca ccatttgttg ccatagcagt 1620

1680

acaattgact gttcctgaat atcttcattt caaggaagaa cttgtggatg gagagtaagc 1740

tctttcttat ttcaatacga aacataaaaa tttacagaag ttgaataatt aacaaatttg 1800

ttgattttta atgtatgcca ggggtaataa tcactttgtg cttgagctgg attttgagcc 1860

atttaatgca tcagttcctc gtccatctcg atcgtcatcc attggcaatg gagtccaatt 1920

cctcaatcgt catctttcct caattatgtt tcgcagcaaa gactctctgg accccttact 1980

tgatttcctt agaggacact gtcataaagg gaatgtaagt accaaaagca gttttccctt 2040

tgtaaatgtc tgcttgtccc tgattatcta ctaaatcttt caacacgcgc aaccattata 2100

agaaatgtac aatacttcta gttagaattt catcatcgac aaactatctg ctttactttt 2160

tatttttccc atttgatgga tgatagttta gtttatataa cagatgatat tttggttgaa 2220

gggtaccatg aactttttca caaccactta atggatacat agttgtaata gttgacattt 2280

tggaataata ttgtctcact tggaaatgtt taagaagtat tactacttct atttgtaaga 2340

tggattgttt atctatgcag gtcttgatgt tgaatgatcg tatacagcga atctccaggc 2400

tggagtctgc tctttctaaa gcagaggatt atctctccaa gctatcacca gatacatcct 2460

ataatgagtt cgaatacgcg tgagcttgta cacatttgtt ttgttttctt tcaagcatat 2520

gtaatttctc aagaaaaggg aaatctatag gagttgaaac attctttatg gaaccatgtg 2580

catgcagatt gcaagaaatg ggctttgaga gaggttgggg tgatactgcc agacgtgttt 2640

tggagacgat gcatcttctt tctgacattc ttcaggctcc ggatccatca accttggaga 2700

catttcttgg tagactacct atggtgttca atgtcgtcat attatcccct catggatatt 2760

ttggccaagc aaatgtcttg ggtttgcccg acactggtgg ccaggtaata acaaggagaa 2820

tgaggtcttg tattatgtac tccctccgtt ccaatctata tgaacctatt tgactgggta 2880

tggaaagaaa tgaagacttg taaaacttgt ggttctttag aaattccaaa cattacattt 2940

ggttttttcc ctcttcctgg aaattatact actgaatcat ctctagatgt tccagtttaa 3000

cttgagacgt aagggtaaat aacggaccat tactctgtcc tttcttgcag taggcttggt 3060

acaatgaata tagttcgcat agttgccgga agctagagct gtgttagaaa actcaggaac 3120

attaatttgg cgatgctaat cactgctaat gttactgaag catccatggt tttccttgat 3180

gttattctcc ttttggttgc ttcacaggtt gtctatatac tggatcaagt gcgtgccttg 3240

gaggccgaaa tgcttcttag aataaagcaa caaggactta acttcaagcc tagaatcctt 3300

gtcgtgagta catatatatt atgcaagctc ttatttggtt tgtgggattg cagttgacat 3360

caatttgctt actctgatta ctaaaggtca cacggctgat acctgatgct aaaggaacca 3420

tgtgcaacca gaggttggag aggattagtg gaactgaata ctcgcatatt ttacgtgtcc 3480

cttttaggac agagaaggga atccttcata aatggatatc taggtttgat gtatggcctt 3540

acctggagaa gttcactgag gtaacctctt tgtcccttgg aaattgcctt ttgttgctga 3600

cacttgctag cgtttgcaat 3660

3720

taaaagttta tggctagtgg aaacatcagt cattcatgta ccttatttct atgcccaagt 3780

tgtttaagtt gaaagtaatt tggccaacta tgcaaattgg gagaacgtgt agccaactat 3840

3900

3960

4020

atgtcttctt gaccaggatg tggcaagtga aatgaccgct gagctccagg gaaagccaga 4080

tctgattatt ggcaactaca gtgatggaaa tttagttgcc tcccttttgg catataaaat 4140

gggtgtcaca caggtaggaa atacatgatt ctttatcttg ctagcactaa gtcttgaggt 4200

4260

tgtaccattg ctcatgcctt ggaaaaaaca aagtatcctg attctgacat ctactggaaa 4320

aagtttgagg agaaatatca ttttcatgt cagtttactg ctgatctact ggcaatgaat 4380

4440

atctggtcgt ttaaatctga tatttcttcc ctagtagtct attcaatccg aatttcagtt 4500

4560

4620

gctagaattt ggttctttat ctatcaagta gcataatact acaactattt tttatgtgtg 4680

gcaatttgca atttcaattt tctatttcta taagttgcag cttttcttcc tgttctgatc 4740

atatttacat ggctgaaact caatagaaaa ctaggctagt tgatcaaaag tagttggatg 4800

ctttaaaatt agtagacgtt ttgctaaatg agtgaccaat gttattaaaa aaacgttcat 4860

gttttcaacc cttttggcat acatttgacc actgcccaag attttggata agtacatgca 4920

gtgcttataa ttataaagca ttttatccca ccttgttttt cattatgaaa attaagtaat 4980

ttacgagtat ttgtataagt tacttcataa attagaagta aatctggatt gtgtaaagtt 5040

attcgccccg tatatactga aagctacttg aacaagcaaa aaaacagaca aacgtaacat 5100

tctccatgga ttaatgagac ttgtatatat atatatatat atatgtaaag agagagagag 5160

agagatttgg cttgtaacca catgtatatt atgccatatg gatgtgacat tgatgtgact 5220

agacctaaat gttttgtttc aatgtccacg ggagttttac gtagagttaa gaggagaaga 5280

gagtgaggaa tactaatgtt tgatggtacc ccttggcttc ttgacctgga tactcagtgt 5340

tcttattcat gcctatactt tggtccttga tttcattctc ccttttctag cttgagctgc 5400

atcaaagaaa ttccactgta aaaaaaataa tgctcaccat attggtgcaa catggcaaac 5460

atgtatccta tttgatgatc aatcaacttt atttttctcc tgttaattga cctcagtgtg 5520

taactctcta tgtatgatag cattgtaact tgtgtcatga ttcataaata gggtactaga 5580

attggatggt tgacatagta aatggtcaat tgatgatcca caaaatatgc acctactgat 5640

taaaatgtga tagggcaggt ttatttttgt ttgtggttaa cacagtactt aaccctatat 5700

5760

acaacaatat tgtctttctg agttctattc tgttgttgct tacactttta ttattccagt 5820

aacatagatg tgaagacatt agattggttg cttgcaaatt gatagccact tgtttcagga 5880

agaatactgt tggtcagtac gagagccata ctgcattcac cctcccggga ctatatcgcg 5940

tcgttcatgg cattgatgtt ttcgatccca aattcaatat agtgtctcct ggagctgaca 6000

tgacaattta tttcccatat tctgacaagg aaaaaagact aacgtctttg catggctcga 6060

6120

6180

gatatgattt tctatagata aactaccaat gatattattt aaaaactcct ggatactgta 6240

ttaggagaag aagagaacca ggggtagatg gcattagaat cccttaaatc ttgaagagtc 6300

gtcactaacg ctcccaacac ttctgcctca gaccctcaac taaatactat tattgttgat 6360

ttctttggag aagctataag aatctctctc tccttatggt gaaaatttta cttggcttta 6420

tacttaactt ccaaggctcc ctcttataaa atgcaaaaac tgtctgtatt cactctcttg 6480

6540

tcgtttgagg ataaggagta gaatctgaga agatagacta gtaggtaacc ttagggacgg 6600

atgtggaaat taacatatgg gctcagcttt tctgccgagt gcagaccatg tatatgcgtt 6660

6720

gtagaatctc gaactcgaac cgataaagtt caaatccagg atccgctttt aggtaaactc 6780

taccttggga agtgttatat atatgtccct gattatttct ttttccgttt cctttctatt 6840

ttaattttta aagttatttt tagatggttt tattttttga taagtggtaa gttgttaata 6900

ttccaaatta aatgccattg tcataactat atacatttat aaagaatgat tgatcctagt 6960

ttctcattcc taagatccaa ataaggcaat aaacaatgtc ttagtaattg gacctgcttc 7020

tggtgatcaa cgcttgatcg cgtagttagt tatagatgac tgtaaaaact ttaaccattt 7080

taatggtttt gtcaaagaac aaatatcgga catattatag agaatggact attgtacttt 7140

gcttctgatt ggtcatttta ttgtgatccg taaattggct gtgactgatg tcatatcttt 7200

gcttacagag gtaatctgaa tgataaatca aaacccataa ttttttcaat ggcaaggcta 7260

gaccatgtta agaacattac gggactagtt gagtgctatg ctaaaaatgc cacattgagg 7320

gaattggcga accttgttgt agtagctgga tacaacgatg taaagaaatc cagtgataga 7380

gaagaaataa cagaaattga gaagatgcat gctcttatta aggagcataa attggatggg 7440

caattcagat gggtatcagc ccaaacaaac cgggcacgta atggtgagct ctatcgctat 7500

atagctgacc agagaggtat atttgttcag gtatgctatt tgtattgtat tagtccaatt 7560

7620

ttttatgaag catttggact aacggtggtt gaagctatga cttgtggtct tccaacattt 7680

gcaacttgcc atggtggtcc taatgagatc attgaacccg gtgtatctgg gttccatatt 7740

gatccttatc atcccgataa agctgctgaa ctcatgtcag aattctttca acgctgcaaa 7800

caagatccta ctcactggga aaaaatatct gcatctggtc tccgaaggat tcttgagagg 7860

tctgtagttg tgtacatgta tagaagatta aagaatgcta ccttgatatt tatttgaatc 7920

aaaaataaca ggaacatctc ttttttgaac atcactcaag ttcttatatt aaataatttt 7980

taggtatacg tggaagattt actccgagag gctgatgact ttatctggcg tatatggttt 8040

ctggaagctt gtttcaaaac ttgagaggcg tgaaactaga cgataccttg agatgttcta 8100

cattctcaaa ttccgcgagt tggtgagtgc cttttagctc cttttcagtt ccaataaact 8160

atatatgtgg tttaagtaag tattaagcat aaacatgtcc gtgcttgggg ctgtcgaaaa 8220

tgctatggac atatcctgag ctaaggattt ttcaagaaaa ttgatgttag ctttactcta 8280

tttacaggca aaatctgtac ctctagcaat tgatgacaag tga 8323

<210> 10

<211> 810

<212> PROTEIN

<213> Nicotiana tabacum

<400> 10

Met Ala Asn Pro Lys Phe Thr Arg Val Pro Ser Met Arg Glu Arg Val

1 5 10 15

Glu Asp Thr Leu Ser Ala His Arg Asn Gln Leu Val Ala Leu Leu Ser

20 25 30

Arg Tyr Val Ala Gln Gly Lys Gly Ile Leu Gln Pro His His Leu Ile

35 40 45

Asp Glu Phe Asn Asn Ala Val Cys Asp Asp Thr Ala Cys Glu Lys Leu

50 55 60

Lys Asp Gly Pro Phe Ser Glu Val Leu Lys Ala Thr Gln Glu Ala Ile

65 70 75 80

Val Leu Pro Pro Phe Val Ala Ile Ala Val Arg Pro Arg Pro Gly Val

85 90 95

Trp Glu Tyr Val Arg Val Asn Val Tyr Asp Leu Ser Val Glu Gln Leu

100 105 110

Thr Val Pro Glu Tyr Leu His Phe Lys Glu Glu Leu Val Asp Gly Glu

115 120 125

Gly Asn Asn His Phe Val Leu Glu Leu Asp Phe Glu Pro Phe Asn Ala

130 135 140

Ser Val Pro Arg Pro Ser Arg Ser Ser Ser Ile Gly Asn Gly Val Gln

145 150 155 160

Phe Leu Asn Arg His Leu Ser Ser Ile Met Phe Arg Ser Lys Asp Ser

165 170 175

Leu Asp Pro Leu Leu Asp Phe Leu Arg Gly His Cys His Lys Gly Asn

180 185 190

Val Leu Met Leu Asn Asp Arg Ile Gln Arg Ile Ser Arg Leu Glu Ser

195 200 205

Ala Leu Ser Lys Ala Glu Asp Tyr Leu Ser Lys Leu Ser Pro Asp Thr

210 215 220

Ser Tyr Asn Glu Phe Glu Tyr Ala Leu Gln Glu Met Gly Phe Glu Arg

225 230 235 240

Gly Trp Gly Asp Thr Ala Arg Arg Val Leu Glu Thr Met His Leu Leu

245 250 255

Ser Asp Ile Leu Gln Ala Pro Asp Pro Ser Thr Leu Glu Thr Phe Leu

260 265 270

Gly Arg Leu Pro Met Val Phe Asn Val Val Ile Leu Ser Pro His Gly

275 280 285

Tyr Phe Gly Glyn Ala Asn Val Leu Gly Leu Pro Asp Thr Gly Gly Gln

290 295 300

Val Val Tyr Ile Leu Asp Gln Val Arg Ala Leu Glu Ala Glu Met Leu

305 310 315 320

Leu Arg Ile Lys Gln Gln Gly Leu Asn Phe Lys Pro Arg Ile Leu Val

325 330 335

Val Thr Arg Leu Ile Pro Asp Ala Lys Gly Thr Met Cys Asn Gln Arg

340 345 350

Leu Glu Arg Ile Ser Gly Thr Glu Tyr Ser His Ile Leu Arg Val Pro

355 360 365

Phe Arg Thr Glu Lys Gly Ile Leu His Lys Trp Ile Ser Arg Phe Asp

370 375 380

Val Trp Pro Tyr Leu Glu Lys Phe Thr Glu Asp Val Ala Ser Glu Met

385 390 395 400

Thr Ala Glu Leu Gln Gly Lys Pro Asp Leu Ile Ile Gly Asn Tyr Ser

405 410 415

Asp Gly Asn Leu Val Ala Ser Leu Leu Ala Tyr Lys Met Gly Val Thr

420 425 430

Gln Cys Thr Ile Ala His Ala Leu Glu Lys Thr Lys Tyr Pro Asp Ser

435 440 445

Asp Ile Tyr Trp Lys Lys Phe Glu Glu Lys Tyr His Phe Ser Cys Gln

450 455 460

Phe Thr Ala Asp Leu Leu Ala Met Asn Asn Ser Asp Phe Ile Ile Thr

465 470 475 480

Ser Thr Tyr Gln Glu Ile Ala Gly Thr Lys Asn Thr Val Gly Gln Tyr

485 490 495

Glu Ser His Thr Ala Phe Thr Leu Pro Gly Leu Tyr Arg Val Val His

500 505 510

Gly Ile Asp Val Phe Asp Pro Lys Phe Asn Ile Val Ser Pro Gly Ala

515 520 525

Asp Met Thr Ile Tyr Phe Pro Tyr Ser Asp Lys Glu Lys Arg Leu Thr

530 535 540

Ser Leu His Gly Ser Ile Glu Lys Leu Leu Phe Asp Pro Ala Gln Asn

545 550 555 560

Glu Glu His Ile Gly Asn Leu Asn Asp Lys Ser Lys Pro Ile Ile Phe

565 570 575

Ser Met Ala Arg Leu Asp His Val Lys Asn Ile Thr Gly Leu Val Glu

580 585 590

Cys Tyr Ala Lys Asn Ala Thr Leu Arg Glu Leu Ala Asn Leu Val Val

595 600 605

Val Ala Gly Tyr Asn Asp Val Lys Lys Ser Ser Asp Arg Glu Glu Ile

610 615 620

Thr Glu Ile Glu Lys Met His Ala Leu Ile Lys Glu His Lys Leu Asp

625 630 635 640

Gly Gln Phe Arg Trp Val Ser Ala Gln Thr Asn Arg Ala Arg Asn Gly

645 650 655

Glu Leu Tyr Arg Tyr Ile Ala Asp Gln Arg Gly Ile Phe Val Gln Pro

660 665 670

Ala Phe Tyr Glu Ala Phe Gly Leu Thr Val Val Glu Ala Met Thr Cys

675 680 685

Gly Leu Pro Thr Phe Ala Thr Cys His Gly Gly Pro Asn Glu Ile Ile

690 695 700

Glu Pro Gly Val Ser Gly Phe His Ile Asp Pro Tyr His Pro Asp Lys

705 710 715 720

Ala Ala Glu Leu Met Ser Glu Phe Phe Gln Arg Cys Lys Gln Asp Pro

725 730 735

Thr His Trp Glu Lys Ile Ser Ala Ser Gly Leu Arg Arg Ile Leu Glu

740 745 750

Arg Tyr Thr Trp Lys Ile Tyr Ser Glu Arg Leu Met Thr Leu Ser Gly

755 760 765

Val Tyr Gly Phe Trp Lys Leu Val Ser Lys Leu Glu Arg Arg Glu Thr

770 775 780

Arg Arg Tyr Leu Glu Met Phe Tyr Ile Leu Lys Phe Arg Glu Leu Ala

785 790 795 800

Lys Ser Val Pro Leu Ala Ile Asp Asp Lys

805 810

<210> 11

<211> 3604

<212> DNA

<213> Nicotiana tabacum

<400> 11

atgtttacat ggctgaaact caatataaaa aacaagggta ggtgatcaaa aatcgttgga 60

120

ttttcaaccc ttttggcata catttgagca ttgcccaaga ttttggataa gtagatgcag 180

tgcttataat tttaaagcat tgtatcctgc cttgtttttc attgtcaaaa ttaattaact 240

tacaagtatt tctataagtt gcttcataaa ttagaagtaa atctggattg tgtaatgtta 300

ttcgcctcgt aaatactgaa agctgcttga acaagtgaaa aaacacagac aaacgtaaca 360

ttctccatgg attgatgaga cttgtaaaat acatatatag aaatttggct tgtaaccaca 420

tgtatattat gccatatgga tgtgacattg atgtgactag acctaaatgt tttgtttcca 480

tgtccactgg agttttacgt atagttaaga ggagaaaaga ctgaggaata ctaatgtatg 540

atggtacccc tttgcttctt gacctggata cccagtgttc ctattcatgc ctatactttg 600

gtccttgatt tcactctccc ttttctaact tgagctgcat caaagaaatt tccactgtaa 660

aaaaataaat aatgctcacc atatctctgc aacattgcaa acatgtatcc catatgattg 720

atattggtgc gacatggcaa acatgtatcc tatttgatga tcaatcaaat ttatttttcc 780

840

catgattcat gaatagggta ctagaattgc atggttgaca aatattaact ggtcgattga 900

tgatccacaa aacatgcact tactgactaa aatgtgatgg gacagattta ttttgtttg 960

tgattaacac agtacttaac cctatactta atacaatttg gcctagctac aatcttttct 1020

tcagtgcaaa ttccttgtta cacgaccaat attgtctttc tgagttctat tctgttgtta 1080

cttacacttt tattattcga ataagacatt agattgcttg catgcaaatt gatagccact 1140

tgtttcagga agaatactgt tggtcagtac gagagccata ctgcattcac cctcccagga 1200

ctatatcgcg tcgttcatgg cattgatgtt ttcgatccca aattcaatat agtgtctcct 1260

ggagctgaca tgacaattta cttcccatat tctgacaagg aaaaaagact aacgtctttg 1320

catggctcga ttgagaagtt gttatttgat cctgcgcaga atgaagagca tatgtaagtg 1380

acatccatt gtacttattt taatttggaa tagatgacat acttatttgc atgaatataa 1440

actgacaacc cagagatttc ctacattaga aaaggagggt ctgatatgat tttctacaaa 1500

taaattccca gtgatattgt tcaaaaagtc ctggatactt tattatgaga gaaccaggga 1560

tagatggcac tagaatccct taatcttgag aagtcgccac ttatcgctcc caacactttc 1620

tgagaccctc aagtaactac tattattgtt tgatatcttg gagaagctat aagaatcttt 1680

ttctccttat tgtaattttt tttacgtgac tttaaactta acttccaagc tccttctgat 1740

aaaatgcaaa aactgtctgt attcactgtc ttggtttatt aacaattgat ccaatcaaat 1800

1860

tctgagaaga tagactagta ggtaacctta ggggcggatg tagaaatcaa cgtatgggtt 1920

cagctttgtt gcagaccctg tatatgcatt aaaaaaatca ctaaataagt aaataattga 1980

ttttgaaccc agtaaatcaa aatgagttgt agtagaatcc tgaactcgaa ccgataaagt 2040

tggatccact accgggtaaa ctctaccttg agaagtgttt atatatgtcc ctaattattt 2100

2160

gacaagtggt aagttgttag tattccaaat taaatgccat tgccataact atatacattt 2220

2280

2340

tgactgtaaa aaccttaatc attttaatgg ttttgtcaaa gaacaaatat cggacatatt 2400

atagcgaatg gactattgta cttttcttct gattggtcat tttattgtga tccgtaagtt 2460

ggctgagact gatgtcatat ctttgcttac agaggtaatc tgaatgataa atcaaaaccc 2520

ataatttttt caatggcaag gctagaccat gttaagaaca ttacgggact agttgagtgc 2580

tatgctaaaa atgccacatt gagggaattg gctaaccttg ttgttgtagc tggatacaac 2640

gatgtaaaga aatccagtga tagagaagaa atagcagaaa ttgagaagat gcatgctctt 2700

attaaggagc ataaattgga tgggcaattc agatggatag cagcccaaac aaaccgggca 2760

cgtaatggtg agctctatcg ctatatagct gacaagagag gtatatttgt tcaggtacgc 2820

tgtttgtatt gtatttgtcc acattccttt ttttgcaccg aaagaaaggt tgttattgtg 2880

acaaatatgt ttgttttagc ctgcatttta tgaagcattt ggactcacgg tggttgaagc 2940

tatgacttgt ggtcttccaa catttgcaac ttgccatggt ggtccgaacg agatcattga 3000

acacggtgta tctgggttcc atattgatcc ttatcatccc gataaagctg ctgaactcat 3060

ggcagaattc tttcaacgct gcaaacaaga tcctactcac tgggaaaaaa tatctgcatc 3120

3180

tgttcccttg atattatttg aatgaaaaat aacagtaaca tctctttttg aacatcgctc 3240

aagttcttgt gttaaataat tgttaggtat acgtggaaaa tttactccga gaggctgatg 3300

actttgtctg gtgtatatgg tttctggaag cttgtttcaa aacttgagag gcgcgaaact 3360

agacgatacc ttgagatgtt ctacattctc aaattccgcg agttggtgag tgcctttttg 3420

ctcattttca gttacaatca actatatatg tggtttaaat acgtattaag cataaacatg 3480

tccgtgattg cggctgtcga aaatgctatg gacatatcct gagctaagga gttttcaaga 3540

gaattgattt ggcttactct gtttacaggc aaaatctgtt cctctggcaa ttgatgacaa 3600

gtga 3604

<210> 12

<211> 335

<212> PROTEIN

<213> Nicotiana tabacum

<400> 12

Met Phe Thr Trp Leu Lys Leu Asn Ile Lys Asn Lys Gly Arg Lys Asn

1 5 10 15

Thr Val Gly Gln Tyr Glu Ser His Thr Ala Phe Thr Leu Pro Gly Leu

20 25 30

Tyr Arg Val Val His Gly Ile Asp Val Phe Asp Pro Lys Phe Asn Ile

35 40 45

Val Ser Pro Gly Ala Asp Met Thr Ile Tyr Phe Pro Tyr Ser Asp Lys

50 55 60

Glu Lys Arg Leu Thr Ser Leu His Gly Ser Ile Glu Lys Leu Leu Phe

65 70 75 80

Asp Pro Ala Gln Asn Glu Glu His Ile Gly Asn Leu Asn Asp Lys Ser

85 90 95

Lys Pro Ile Ile Phe Ser Met Ala Arg Leu Asp His Val Lys Asn Ile

100 105 110

Thr Gly Leu Val Glu Cys Tyr Ala Lys Asn Ala Thr Leu Arg Glu Leu

115 120 125

Ala Asn Leu Val Val Val Ala Gly Tyr Asn Asp Val Lys Lys Ser Ser

130 135 140

Asp Arg Glu Glu Ile Ala Glu Ile Glu Lys Met His Ala Leu Ile Lys

145 150 155 160

Glu His Lys Leu Asp Gly Gln Phe Arg Trp Ile Ala Ala Gln Thr Asn

165 170 175

Arg Ala Arg Asn Gly Glu Leu Tyr Arg Tyr Ile Ala Asp Lys Arg Gly

180 185 190

Ile Phe Val Gln Pro Ala Phe Tyr Glu Ala Phe Gly Leu Thr Val Val

195 200 205

Glu Ala Met Thr Cys Gly Leu Pro Thr Phe Ala Thr Cys His Gly Gly

210 215 220

Pro Asn Glu Ile Ile Glu His Gly Val Ser Gly Phe His Ile Asp Pro

225 230 235 240

Tyr His Pro Asp Lys Ala Ala Glu Leu Met Ala Glu Phe Phe Gln Arg

245 250 255

Cys Lys Gln Asp Pro Thr His Trp Glu Lys Ile Ser Ala Ser Gly Leu

260 265 270

Arg Arg Ile Leu Glu Arg Tyr Thr Trp Lys Ile Tyr Ser Glu Arg Leu

275 280 285

Met Thr Leu Ser Gly Val Tyr Gly Phe Trp Lys Leu Val Ser Lys Leu

290 295 300

Glu Arg Arg Glu Thr Arg Arg Tyr Leu Glu Met Phe Tyr Ile Leu Lys

305 310 315 320

Phe Arg Glu Leu Ala Lys Ser Val Pro Leu Ala Ile Asp Asp Lys

325 330 335

<210> 13

<211> 4030

<212> DNA

<213> Nicotiana tabacum

<400> 13

atggcggaac gtgtgctgac tcgtgttcat agccttcgtg aacgtcttga tgctactttg 60

gctgctcatc gcaatgagat tttgctgttt ctttcaaggt atagtcttag cagattgttc 120

tttgatttag ttgttattgc cagttctaat gtatgggctt atatataaac aaagtgttga 180

agtatgcaac catataaact gacagcttaa aatgcttgag agaacacact tttatttatt 240

taattatgcc ttcagcacaa gaagtggaac ttgacgcaat ggaaccatag gtcacggggtt 300

caagtcttgg aacagcctgc aatctaaggc tgcgtgtagt agaccctagt ggtccggccc 360

ttccacatat ctcgcttagt gtaccgggcc cattgagtac gggttcggcc gaacccagtc 420

gctttggtcc aatccatata tttgtcttaa aaatatattg aatatataca aattgttaat 480

540

agccatggaa aagggatact gaaacctcac cagttgctgg ctgaatttga ttcaattcac 600

aaagaagaca aaaacaaact gaatgatcat gcttttgaag aagtcctgaa atccactcag 660

gtatttgtgg ttttagtgtt aggtgatgga tagcatttat tgttttacta agatcacata 720

tgtgtcagtt tgtggctagt atttaaaatc tggtgtattt tgtcatacta ggaagcaatt 780

gttttgtccc cttgggttgc gcttgccatt cgtctgaggc ctggtgtgtg ggaatacgtt 840

cgtgtgaatg tcaacgctct tgttgttgag gagcttaccg tgcctgagta tttgcaattc 900

aaggaagaac ttgttaatgg aacgtaagtt ttaggttcga atttgttgat ttgttagata 960

acatgttctg aactttttga ttaaagttgt gtttttgact gatgcagctc gcacgataac 1020

tttgttcttg agttggattt tgagcccttc actgcatcat ttccaaaacc aaccctcacc 1080

aaatcaattg gaaatggagt tgaattcctt aaccgacacc tctctgccaa aatgttccat 1140

gacaaggaaa gcatgacccc tcttctcgag tttcttcgag ttcaccacta caagggcaag 1200

gtaaacttgt ttttcctgtt tgtctatgaa tttagtttag ttgttttgct ccgcgaaaat 1260

ttcagtggaa actgattat gcaaccactg agtgattaat atgttcaaac ttaccgactt 1320

ctggttttct gtgtagacaa tgatgctgaa tgacagaatt caggacttaa atactctcca 1380

aaatgtccta aggaaagctg aggaatacct cactaccctt tcccctgaaa cttcatactc 1440

ggcatttgag cacaagttcc aagaaattgg cttggagagg ggttggggtg acactgcgga 1500

gcgtgttcta gagatgatct gcatgctcct ggatctcctc gaggctcctg actcgtgcac 1560

gcttgagaag ttccttggta gaattccaat ggtttttaat gtggtcatac tttcacccca 1620

tggttatttc gccggaaa atgtcttggg ttaccccgac actggtggcc aggtgcactg 1680

cttatctgtg ttcggtctta ttatctcttt aaaccctact gccacaagtg ctgagatgaa 1740

cctcctttaa tttgcaggtt gtctatattt tggatcaagt tcctgctttg gagcgtgaga 1800

tgctcaagcg cataaaggag caaggacttg acatcaaacc gcgtattctt attgttcgta 1860

ttcccagtaa ttgtgtttaa acttatgatt atgcaggatt ttatctgttc taatacagca 1920

ctcttgctta aattctcagg ttactcggct gctgcctgat gcggttggta ccacttgtgg 1980

tcagaggctt gagaaagtgt ttggaacaga gcactcacac attcttaggg tcccctttag 2040

gaccgagaag ggcattgttc gcaaatggat ctctcgcttt gaagtctggc catacatgga 2100

gacattcact gaggtgaagc aagctttctc tattcatttt tcaatcttcc aattggtttt 2160

ggcagcaatt ttctgcttgc tttgacttcc gctaaaactt cggattttat tgcattagga 2220

tgtggcgaaa gaaattgctg cagaattgca ggctaagcca gatcttatca ttggcaatta 2280

tagtgagggc aaccttgctg cctccttgtt ggctcacaaa ttaggtgtaa cacaggtcgg 2340

2400

aagagttctc tctctctttt ttttttccgt ctactttgcc ttatgcagtg cacgatagct 2460

catgctttgg agaaaacaaa atatcctgat tctgatatct acttgaagaa atttgatgaa 2520

aaataccatt tctcagccca gtttactgcc gatcttattg caatgaatca caccgatttc 2580

atcatcacca gcactttcca ggagatagcg ggaaggtatt tttacatcag tttcccactc 2640

tgattaaatt acaatgtatt tccctatatg attaaatact gtgtttgatc ctaaatcatt 2700

2760

cctggactgt atagagttgt tcacggcatt gatgtgtttg accccaaatt taacattgtg 2820

tcaccaggag ctgatatgaa tctctatttc ccatactacg agaaggaaaa gagattgaca 2880

gcatatcacc ctgaaattga ggagctgctg tttagtgatg ttgagaatga cgaacacatg 2940

3000

3060

ttcactatgg ctagattggga tcgagtgaag aacttaactg gacttgtcga gctgtacgcc 3120

aagaacccac ggctaaggga gttggttaac cttgtcgtgg ttggaggaga ccgaaggaaa 3180

gaatccaaag acttggaaga acaggcagag atgaagaaga tgtacgaact tataaagact 3240

cacaatttga acggccaatt ccgatggatt tcttcccaga tgaaccgcgt gaggaatggc 3300

3360

gcttttggtt tgactgttgt tgaggccatg acctgtggtt tgcctacatt tgcaactaat 3420

cacggtggtc cagctgagat catcgttcac gggaaatctg gtttccacat tgatccatac 3480

cacggggatc aggcagctga acttctcgct gatttctttg agaaatgtaa gaaagaacct 3540

tcgcactggg aagccatttc cgagggcggc cttaagcgta tacaggagaa gtaagcaaac 3600

3660

acctacttgc tgttgttatt gtttgttgct tccttttcac tgttctttga gctgaaggtc 3720

tatcagaaac agtctctcta ccttcacaag gtaggggtaa gatctgcgtg cacgttaccc 3780

3840

attaattaaa aggtacacat ggcaaatata ctcggatcgg ttgttgacac tggctgctgt 3900

atatggattc tggaagcatg tttccaagct tgatcgtctt gaaattcgcc gttatcttga 3960

aatgttctat gctctcaaat tccgcaagct ggtgagtttc attgctttct gcactcctgc 4020

aattgtatag 4030

<210> 14

<211> 808

<212> PROTEIN

<213> Nicotiana tabacum

<400> 14

Met Ala Glu Arg Val Leu Thr Arg Val His Ser Leu Arg Glu Arg Leu

1 5 10 15

Asp Ala Thr Leu Ala Ala His Arg Asn Glu Ile Leu Leu Phe Leu Ser

20 25 30

Arg Ile Glu Ser His Gly Lys Gly Ile Leu Lys Pro His Gln Leu Leu

35 40 45

Ala Glu Phe Asp Ser Ile His Lys Glu Asp Lys Asn Lys Leu Asn Asp

50 55 60

His Ala Phe Glu Glu Val Leu Lys Ser Thr Gln Glu Ala Ile Val Leu

65 70 75 80

Ser Pro Trp Val Ala Leu Ala Ile Arg Leu Arg Pro Gly Val Trp Glu

85 90 95

Tyr Val Arg Val Asn Val Asn Ala Leu Val Val Glu Glu Leu Thr Val

100 105 110

Pro Glu Tyr Leu Gln Phe Lys Glu Glu Leu Val Asn Gly Thr Ser His

115 120 125

Asp Asn Phe Val Leu Glu Leu Asp Phe Glu Pro Phe Thr Ala Ser Phe

130 135 140

Pro Lys Pro Thr Leu Thr Lys Ser Ile Gly Asn Gly Val Glu Phe Leu

145 150 155 160

Asn Arg His Leu Ser Ala Lys Met Phe His Asp Lys Glu Ser Met Thr

165 170 175

Pro Leu Leu Glu Phe Leu Arg Val His His Tyr Lys Gly Lys Thr Met

180 185 190

Met Leu Asn Asp Arg Ile Gln Asp Leu Asn Thr Leu Gln Asn Val Leu

195 200 205

Arg Lys Ala Glu Glu Tyr Leu Thr Thr Leu Ser Pro Glu Thr Ser Tyr

210 215 220

Ser Ala Phe Glu His Lys Phe Gln Glu Ile Gly Leu Glu Arg Gly Trp

225 230 235 240

Gly Asp Thr Ala Glu Arg Val Leu Glu Met Ile Cys Met Leu Leu Asp

245 250 255

Leu Leu Glu Ala Pro Asp Ser Cys Thr Leu Glu Lys Phe Leu Gly Arg

260 265 270

Ile Pro Met Val Phe Asn Val Val Ile Leu Ser Pro His Gly Tyr Phe

275 280 285

Ala Gln Glu Asn Val Leu Gly Tyr Pro Asp Thr Gly Gly Gln Val Val

290 295 300

Tyr Ile Leu Asp Gln Val Pro Ala Leu Glu Arg Glu Met Leu Lys Arg

305 310 315 320

Ile Lys Glu Gln Gly Leu Asp Ile Lys Pro Arg Ile Leu Ile Val Thr

325 330 335

Arg Leu Leu Pro Asp Ala Val Gly Thr Thr Cys Gly Gln Arg Leu Glu

340 345 350

Lys Val Phe Gly Thr Glu His Ser His Ile Leu Arg Val Pro Phe Arg

355 360 365

Thr Glu Lys Gly Ile Val Arg Lys Trp Ile Ser Arg Phe Glu Val Trp

370 375 380

Pro Tyr Met Glu Thr Phe Thr Glu Asp Val Ala Lys Glu Ile Ala Ala

385 390 395 400

Glu Leu Gln Ala Lys Pro Asp Leu Ile Ile Gly Asn Tyr Ser Glu Gly

405 410 415

Asn Leu Ala Ala Ser Leu Leu Ala His Lys Leu Gly Val Thr Gln Cys

420 425 430

Thr Ile Ala His Ala Leu Glu Lys Thr Lys Tyr Pro Asp Ser Asp Ile

435 440 445

Tyr Leu Lys Lys Phe Asp Glu Lys Tyr His Phe Ser Ala Gln Phe Thr

450 455 460

Ala Asp Leu Ile Ala Met Asn His Thr Asp Phe Ile Ile Thr Ser Thr

465 470 475 480

Phe Gln Glu Ile Ala Gly Ser Lys Asp Thr Val Gly Gln Tyr Glu Ser

485 490 495

His Met Ala Phe Thr Met Pro Gly Leu Tyr Arg Val Val His Gly Ile

500 505 510

Asp Val Phe Asp Pro Lys Phe Asn Ile Val Ser Pro Gly Ala Asp Met

515 520 525

Asn Leu Tyr Phe Pro Tyr Tyr Glu Lys Glu Lys Arg Leu Thr Ala Tyr

530 535 540

His Pro Glu Ile Glu Glu Leu Leu Phe Ser Asp Val Glu Asn Asp Glu

545 550 555 560

His Met Cys Val Leu Lys Asn Arg Asn Lys Pro Ile Ile Phe Thr Met

565 570 575

Ala Arg Leu Asp Arg Val Lys Asn Leu Thr Gly Leu Val Glu Leu Tyr

580 585 590

Ala Lys Asn Pro Arg Leu Arg Glu Leu Val Asn Leu Val Val Val Gly

595 600 605

Gly Asp Arg Arg Lys Glu Ser Lys Asp Leu Glu Glu Gln Ala Glu Met

610 615 620

Lys Lys Met Tyr Glu Leu Ile Lys Thr His Asn Leu Asn Gly Gln Phe

625 630 635 640

Arg Trp Ile Ser Ser Gln Met Asn Arg Val Arg Asn Gly Glu Leu Tyr

645 650 655

Arg Tyr Ile Ala Asp Thr Arg Gly Ala Phe Val Gln Pro Ala Phe Tyr

660 665 670

Glu Ala Phe Gly Leu Thr Val Val Glu Ala Met Thr Cys Gly Leu Pro

675 680 685

Thr Phe Ala Thr Asn His Gly Gly Pro Ala Glu Ile Ile Val His Gly

690 695 700

Lys Ser Gly Phe His Ile Asp Pro Tyr His Gly Asp Gln Ala Ala Glu

705 710 715 720

Leu Leu Ala Asp Phe Phe Glu Lys Cys Lys Lys Glu Pro Ser His Trp

725 730 735

Glu Ala Ile Ser Glu Gly Gly Leu Lys Arg Ile Gln Glu Lys Tyr Thr

740 745 750

Trp Gln Ile Tyr Ser Asp Arg Leu Leu Thr Leu Ala Ala Val Tyr Gly

755 760 765

Phe Trp Lys His Val Ser Lys Leu Asp Arg Leu Glu Ile Arg Arg Tyr

770 775 780

Leu Glu Met Phe Tyr Ala Leu Lys Phe Arg Lys Leu Val Ser Phe Ile

785 790 795 800

Ala Phe Cys Thr Pro Ala Ile Val

805

<210> 15

<211> 4054

<212> DNA

<213> Nicotiana tabacum

<400> 15

atggccgaac gtgtgctaac tcgtgttcac agccttcgcg aacgtcttga tgctactttg 60

gctgctcatc gcaatgagat tttgctgttt ctttcaaggt atagtcttag cagattgttc 120

tttgatttag ttggtgttat ttgccagttc taatgtatgg actaatatat gaacaaagtg 180

cgaccatttc aactgacaac ttaaaatgtt tgagagaata cacgtttatt tacttaatta 240

tggcttgagc ataggaagtg tatcttggcg taactcgtaa agttgacctc atgtgacaag 300

360

caatagatcg cccttccacg gacccgcgca tagcgggaac ttagtgcacc ggttggggctg 420

tccttttttta tgtcttcagc acaaaaattt agtttaaaca tgtgtatcat ggattattca 480

tgctggtttt gccggttgca ggattgaaag ccacggaaaa gggatattga aacctcacca 540

gttgctggct gagtttgaat caattcacaa agaagacaaa aacaaactga atgatcatgc 600

ttttgaagaa gtcctgaaat ctactcaggt aatttgtggt tttagtgtta ggtgatggat 660

agcatttatt gtcttactaa gatcatatat gtgtcagttt gtggctagta tttgaaaagt 720

ctggtgtggt ttgtcatact aggaagcaat tgtcttgtcc ccttgggttg cgcttgccat 780

tcgtctgcgg cctggtgtgt gggaatatgt tcgtgtgaat gtcaatgcac ttattgtcga 840

ggagctgact gtgcctgaat atttgcaatt caaggaagaa cttgttaatg gaacgtaagt 900

tttaggttcg aaatgatgat ttgttaaata atatgttctg aactttttga ttaatgttgt 960

gttttcccct gatgcagctc gaacgataac tttgttcttg agctggattt tgagcccttc 1020

actgcatcat ttcccaaacc aaccctcacc aaatcaattg gaaatggagt tgaattcctc 1080

aaccgacacc tctctgccaa aatgttccat gacaaggaaa gcatgacccc tcttctcgag 1140

tttcttcgag ttcatcacta caagggcaag gtaaacttgt ttttcctgtt tgtctatgaa 1200

tttagtttct gaaagttgct ttgcttcgtg aattttttag tggcaactga tttatgattt 1260

tctgtgcaga caatgatgct gaatgacaga gttcaggact taaacactct ccaaaatgtc 1320

ctaaggaagg ctgaggaata tctcactacc ctttcccctg aaacttcata ctcggtattt 1380

gagcacaagt tccaagaaat tggcctagag aggggctggg gtgacaatgc tgagcgtgtt 1440

ctagagatga tctgcatgct cctggatctc ctcgaggctc cagactcatg cactcttgag 1500

aagttccttg gtagaattcc tatggttttt aatgtggtca ttctttcacc tcacggatat 1560

ttcgcccagg aaaatgtctt gggttacccc gatactggtg gccaggtgca ctgcttattt 1620

gtaacacctt acgcttttcc ctctgaaact tatttgcggc aagttctaag gtcctccttc 1680

cttaatttgc aggttgtcta tattttggat caagttccgg ccttggagcg tgagatgctc 1740

aagcgcataa aggagcaagg acttgatatc aaaccgcgta ttcttattgt tcgtatctcc 1800

aataattgcg tttaaactta tgattgtgca ggatttgatc tgttcaaatc taatgactga 1860

ttttcttttt tttttttttt tccctcaggt tactcggctg ctgcctgatg cggttggtac 1920

cacttgtggt cagcggcttg agaaagtgtt tggaacagag cattcacata ttcttagggt 1980

cccctttagg accgagaagg gcatcgttcg caaatggatc tctcgctttg aagtctggcc 2040

ttacatggag acattcactg aggtgaagca agctttctct attcattttt caatcttcca 2100

atctgttttg gcagcaattt ttcacttact aacactttgg ctttcgctaa aacttcggat 2160

tttattacat taggatgtgg caaaagaaat tgctgcagaa ctgcaggcaa agccagatct 2220

tataatcggc aactacagcg agggcaacct tgctgcctcc ttgttggctc acaagttagg 2280

tgtaactcag gtctgtaatg tttgtcacct gttatttcaa ctttgcattt cctttcattt 2340

gcaactagaa gttaagagtt ctctctcttt tatcttttcc gtctattttg ccttctgcag 2400

tgcaccatag ctcatgcgtt ggagaaaaca aaatatcctg attctgatat ctacttgaag 2460

aaatttgatg aaaaatacca tttctcagcc cagtttactg ccgatcttat tgcaatgaat 2520

cacaccgatt tcataatcac cagcactttc caggagatag cgggaaggta ttacatcaca 2580

atggatttcc gatatgatta aattagttaa tttaatccta cttcattgtg tttgatccta 2640

aaacttttct aaatttccca gcaaggacac tgttggacag tacgagagcc acatggcttt 2700

cacgatgcct ggattgtata gagttgttca cggcattgat gtgttcgatc ccaaattcaa 2760

cattgtgtca ccaggagctg atatgaatct ctatttcccc tacttcgaga aggaaaagcg 2820

attgacagca tatcaccctg aaattgagga gctgctgttt agcgatgttg agaatgacga 2880

acacatgtat gttactaaac tagcaatcct gctgcaaaat tgtggctaat tatgtaaaaa 2940

agtttttact gaatagattt gtgcttctat caggtgtgtg ctgaaggaca ggaataagcc 3000

3060

gtacgccaag aacccggc taagggagtt ggttaacctt gtcgtggttg gtggagaccg 3120

3180

aaagacgcac aatttaaacg gccaattccg atggatttct tcccagatga accgcgtgag 3240

gaatggcgaa ctctacaggt acattgccga tactagggga gcttttgtgc agcctgcatt 3300

3360

aactaatcac ggtggtccag ctgagatcat cgttcacggg aagtctggtt ttcacattga 3420

tccataccac ggcgagcagg cagctgaact tctagctgat ttctttgaga gatgtaagaa 3480

agaaccttca cactgggaag ccatttccga gggcggcctt aagcgtatac aggagaagta 3540

agcaagctgc tactcttttc atttttgcaa aacctaccat gatcattatt aagctcattt 3600

ttgcaaaacc tacttgttat tctttgttgc ttccttttcc ctgttttttg agccgaggtt 3660

ttatcgaaaa catgctttct accttcacaa ggtaggggta aggtctgcgt ttgttattat 3720

tgttgttgtt gattctctgc gaattaatta aaaggtacac atggcaaatc tactcggatc 3780

ggttgttgac actggctgct gtttatggat tctggaagca tgtttccaaa cttgatcgtc 3840

ttgaaattcg tcgttatctt gaaatgttct atgctctaaa attccgcaaa ctggtgagtt 3900

tcactgcttt ctgcactctt ccaattgtta gttgagtgca ctcatttaaa ctgtagctaa 3960

agctgttgta aatcttcagt taagcagctg ctaatgaagt ttttatcttt tgtttttggt 4020

tcaggctgaa gctgtcccgt tggctgttga gtaa 4054

<210> 16

<211> 805

<212> PROTEIN

<213> Nicotiana tabacum

<400> 16

Met Ala Glu Arg Val Leu Thr Arg Val His Ser Leu Arg Glu Arg Leu

1 5 10 15

Asp Ala Thr Leu Ala Ala His Arg Asn Glu Ile Leu Leu Phe Leu Ser

20 25 30

Arg Ile Glu Ser His Gly Lys Gly Ile Leu Lys Pro His Gln Leu Leu

35 40 45

Ala Glu Phe Glu Ser Ile His Lys Glu Asp Lys Asn Lys Leu Asn Asp

50 55 60

His Ala Phe Glu Glu Val Leu Lys Ser Thr Gln Glu Ala Ile Val Leu

65 70 75 80

Ser Pro Trp Val Ala Leu Ala Ile Arg Leu Arg Pro Gly Val Trp Glu

85 90 95

Tyr Val Arg Val Asn Val Asn Ala Leu Ile Val Glu Glu Leu Thr Val

100 105 110

Pro Glu Tyr Leu Gln Phe Lys Glu Glu Leu Val Asn Gly Thr Ser Asn

115 120 125

Asp Asn Phe Val Leu Glu Leu Asp Phe Glu Pro Phe Thr Ala Ser Phe

130 135 140

Pro Lys Pro Thr Leu Thr Lys Ser Ile Gly Asn Gly Val Glu Phe Leu

145 150 155 160

Asn Arg His Leu Ser Ala Lys Met Phe His Asp Lys Glu Ser Met Thr

165 170 175

Pro Leu Leu Glu Phe Leu Arg Val His His Tyr Lys Gly Lys Thr Met

180 185 190

Met Leu Asn Asp Arg Val Gln Asp Leu Asn Thr Leu Gln Asn Val Leu

195 200 205

Arg Lys Ala Glu Glu Tyr Leu Thr Thr Leu Ser Pro Glu Thr Ser Tyr

210 215 220

Ser Val Phe Glu His Lys Phe Gln Glu Ile Gly Leu Glu Arg Gly Trp

225 230 235 240

Gly Asp Asn Ala Glu Arg Val Leu Glu Met Ile Cys Met Leu Leu Asp

245 250 255

Leu Leu Glu Ala Pro Asp Ser Cys Thr Leu Glu Lys Phe Leu Gly Arg

260 265 270

Ile Pro Met Val Phe Asn Val Val Ile Leu Ser Pro His Gly Tyr Phe

275 280 285

Ala Gln Glu Asn Val Leu Gly Tyr Pro Asp Thr Gly Gly Gln Val Val

290 295 300

Tyr Ile Leu Asp Gln Val Pro Ala Leu Glu Arg Glu Met Leu Lys Arg

305 310 315 320

Ile Lys Glu Gln Gly Leu Asp Ile Lys Pro Arg Ile Leu Ile Val Thr

325 330 335

Arg Leu Leu Pro Asp Ala Val Gly Thr Thr Cys Gly Gln Arg Leu Glu

340 345 350

Lys Val Phe Gly Thr Glu His Ser His Ile Leu Arg Val Pro Phe Arg

355 360 365

Thr Glu Lys Gly Ile Val Arg Lys Trp Ile Ser Arg Phe Glu Val Trp

370 375 380

Pro Tyr Met Glu Thr Phe Thr Glu Asp Val Ala Lys Glu Ile Ala Ala

385 390 395 400

Glu Leu Gln Ala Lys Pro Asp Leu Ile Ile Gly Asn Tyr Ser Glu Gly

405 410 415

Asn Leu Ala Ala Ser Leu Leu Ala His Lys Leu Gly Val Thr Gln Cys

420 425 430

Thr Ile Ala His Ala Leu Glu Lys Thr Lys Tyr Pro Asp Ser Asp Ile

435 440 445

Tyr Leu Lys Lys Phe Asp Glu Lys Tyr His Phe Ser Ala Gln Phe Thr

450 455 460

Ala Asp Leu Ile Ala Met Asn His Thr Asp Phe Ile Ile Thr Ser Thr

465 470 475 480

Phe Gln Glu Ile Ala Gly Ser Lys Asp Thr Val Gly Gln Tyr Glu Ser

485 490 495

His Met Ala Phe Thr Met Pro Gly Leu Tyr Arg Val Val His Gly Ile

500 505 510

Asp Val Phe Asp Pro Lys Phe Asn Ile Val Ser Pro Gly Ala Asp Met

515 520 525

Asn Leu Tyr Phe Pro Tyr Phe Glu Lys Glu Lys Arg Leu Thr Ala Tyr

530 535 540

His Pro Glu Ile Glu Glu Leu Leu Phe Ser Asp Val Glu Asn Asp Glu

545 550 555 560

His Met Cys Val Leu Lys Asp Arg Asn Lys Pro Ile Ile Phe Thr Met

565 570 575

Ala Arg Leu Asp Arg Val Lys Asn Leu Thr Gly Leu Val Glu Leu Tyr

580 585 590

Ala Lys Asn Pro Arg Leu Arg Glu Leu Val Asn Leu Val Val Val Gly

595 600 605

Gly Asp Arg Arg Lys Glu Ser Lys Asp Leu Glu Glu Gln Ala Glu Met

610 615 620

Lys Lys Met Tyr Glu Leu Ile Lys Thr His Asn Leu Asn Gly Gln Phe

625 630 635 640

Arg Trp Ile Ser Ser Gln Met Asn Arg Val Arg Asn Gly Glu Leu Tyr

645 650 655

Arg Tyr Ile Ala Asp Thr Arg Gly Ala Phe Val Gln Pro Ala Phe Tyr

660 665 670

Glu Ala Phe Gly Leu Thr Val Val Glu Ala Met Thr Cys Gly Leu Pro

675 680 685

Thr Phe Ala Thr Asn His Gly Gly Pro Ala Glu Ile Ile Val His Gly

690 695 700

Lys Ser Gly Phe His Ile Asp Pro Tyr His Gly Glu Gln Ala Ala Glu

705 710 715 720

Leu Leu Ala Asp Phe Phe Glu Arg Cys Lys Lys Glu Pro Ser His Trp

725 730 735

Glu Ala Ile Ser Glu Gly Gly Leu Lys Arg Ile Gln Glu Lys Tyr Thr

740 745 750

Trp Gln Ile Tyr Ser Asp Arg Leu Leu Thr Leu Ala Ala Val Tyr Gly

755 760 765

Phe Trp Lys His Val Ser Lys Leu Asp Arg Leu Glu Ile Arg Arg Tyr

770 775 780

Leu Glu Met Phe Tyr Ala Leu Lys Phe Arg Lys Leu Ala Glu Ala Val

785 790 795 800

Pro Leu Ala Val Glu

805

<210> 17

<211> 3752

<212> DNA

<213> Nicotiana tabacum

<400> 17

atggcctcaa cagttgctga tagcatgcct gatgctttga aacaaagccg gtatcatatg 60

120

gtcaaatttc catgcataat gctaactact tttcttcttt ttgacttcaa aattggatgt 180

gaaaggttca ttgcaatggg aaggaggcta atgaagttga aacatttaac agaagaaata 240

gaagaaacta ttgaagacaa ggcagaaaga accaggattt tggagggttc acttggaaaa 300

attatgagtt ccacacaggt cagcaccatt taaccaactt agttgaacag gaaaaaaaga 360

aaaagcaaaa gagttattgc aaggcgtaac gattttcttt gaaattttca ggaggcagct 420

gttgttccac cttatgttgc ttttgcagta aggcacaatc ctggcttctg ggattatgtc 480

aaagttaacg ctgaaactct ctctgtggaa gctatttcag ccagggaata tctcaaattc 540

aaagagatga tctttgacga agactggtaa gtggaaaatt gtatcatttt aaagagaaac 600

aattttgtaa catacaagaa tagttttgat ggttgaatgt gcaagcaggg caaaggatga 660

taatgcactg gaagtagatt ttggtgcttt tgactactct aatcctcggt tagccctttc 720

ctcttctgtc ggaaatggggc tcaactttat ctcaaaagtt ctgtcttcaa agtttggtgg 780

aaagccagag gacgcccagc ctttgcttga ttacttacta gctcttaatc atcaaggaga 840

ggtatgaaaa tggactacct ttgtttctta aaggtattat ataatgatgc gcgttataaa 900

gttccttttt aaattgaaac tttgcagaat ctaatgatca atgagaatct gaatggtgtt 960

gctaagcttc aagcagcatt gatagtagct gaagtttttg tatcttcctt tcccaaagac 1020

acaccttata aagactttga gcataagtaa gcttctcata tgcttccatt gtcatatgca 1080

gtataccaat gacatgctac cgaaaagttg tttatgtttg tgacttgatt atgaaaactc 1140

taggctcaaa gaatggggct ttgataaagg gtggggtcac aatgcaggaa gagtaagaga 1200

gacaatgaga ctgctttccg agataatcca agcaccagat cccataaata tggagtcctt 1260

tttcagcaag cttcctacta cattcaacat tgttatcttc tccattcatg gttactttgg 1320

ccaagcagat gtccttggtc tgcccgatac tggaggccag gtctacatat acagcaattt 1380

atctcctttt gcctcatatt gcttattagc gacacttgca tcattgaaat cagactttta 1440

cttcacaggt tgtttatatt ctggatcaag taagggcttt agaggaggaa atgttacaaa 1500

gaatcaagca gcaagggcta aacgtgaagc ccaagattct tgtggtgagt tttgcaaaaa 1560

1620

ttcctgcttt catgatgtaa acaggtatct cgtctcatac cagatgctcg agggacaaca 1680

tgcaatcagg agatggaacc tattcttaac tcatcccatt ctcacatcct gagaattcca 1740

ttcaggactg agaaaggagt tcttcgccaa tgggtttctc ggtttgatat ctatccttac 1800

ttggagaact atgccaaggc aagtcttcta acaaaattac cacctattca tacactttat 1860

ttactttctt gaactaatcg tttggtttgt gacgtatatc attaggatgc ttctgctaag 1920

atacttgagc tcatggaagg taaaccagac ctcataattg ggaactacac tgatggaaat 1980

ttagtggcat ctctattggc caacaaactt ggagttactc aggttccgta gctgatcata 2040

tgatcatatt ttctacattg tttcttgata attaaatgga aatcttattg gatgataaca 2100

ttttagggaa ccattgctca tgcattagag aaaactaagt atgaagattc tgatgtgaag 2160

tggaagcagt ttgatcccaa gtaccacttt tcttgccaat ttactgccga tttattggca 2220

atgaatgctg ctgattttat cattaccagc acatatcaag aaatcgctgg aaggttagca 2280

ctgactctct cagtatattt ggcaacttaa tgaatttact gcagtggcca acactaaaag 2340

ctatcattcg tccttcagcg aaactaggcc tggacaatat gaaagtcaca cagcatttac 2400

catgccgggg ctttatagag ctgtttcagg catcaatgta tttgatccaa agttcaacat 2460

tgctgctcct ggggctgaac agtctaccta tttccctttc actgagaaac agaaacgatt 2520

cagcacattt cgtcctgcta ttaacgaatt actttacagt aatgaggaaa acaatgagca 2580

catgtaagtc taattgccca ttttcctaat ctaaccattg cttaaatcgt tctgttttta 2640

ccggatgtgt ggtacttatc agtaacattt ttttttggat cagtggattt cttgcagacc 2700

ggaaaaaacc aattatattt tcaatggcga gatttgatac agtgaagaac ctgtcaggct 2760

tgactgagtg gtatgggaag aataagaagt tgcggaactt ggtaaacctt gttattgttg 2820

ggggattctt cgatccatca aaatcaaaag accgggagga agcagctgaa atcaagaaga 2880

tgcatgaatt gattgagaaa taccagctca agggacaaat gagatggata gcagctcaaa 2940

ctgataaata tcgaaatagt gagctatacc gaactattgc tgacactaag ggagcttttg 3000

tccaaccggc tttatatgaa gcttttggac taaccgttat tgaagcaatg gattgtggat 3060

tgcctacgtt tgcaactaat caaggtggac ctgcagaaat cattgttgat ggggtttcag 3120

gtttccatat tgatccttac aatggggacg aatcaagcaa gaaaatagct gatttctttg 3180

agaagtgtaa ggttgattct aaatattgga acaggatatc tgagggaggt ctcaagcgca 3240

ttgaagaatg gtaacaaact agttccaagt ttaaaaaatg gaaaaaatgc ttatcatgtt 3300

3360

3420

3480

3540

atgctacttt tgtaatttca caggccaaaa gcgtgccgat cccacatgaa gagccattgc 3600

cacttgcaac atcagactct actcaatccc aagaattaaa actaccacta ccagttccag 3660

cagcagtagc taaagttctg ccattaacaa ggcatgcttt taacttaatt acttctctac 3720

ctagagtaac tggtaaagtg gatgtcaagt ga 3752

<210> 18

<211> 840

<212> PROTEIN

<213> Nicotiana tabacum

<400> 18

Met Ala Ser Thr Val Ala Asp Ser Met Pro Asp Ala Leu Lys Gln Ser

1 5 10 15

Arg Tyr His Met Lys Arg Cys Phe Ala Arg Phe Ile Ala Met Gly Arg

20 25 30

Arg Leu Met Lys Leu Lys His Leu Thr Glu Glu Ile Glu Glu Thr Ile

35 40 45

Glu Asp Lys Ala Glu Arg Thr Arg Ile Leu Glu Gly Ser Leu Gly Lys

50 55 60

Ile Met Ser Ser Thr Gln Glu Ala Ala Val Val Pro Pro Tyr Val Ala

65 70 75 80

Phe Ala Val Arg His Asn Pro Gly Phe Trp Asp Tyr Val Lys Val Asn

85 90 95

Ala Glu Thr Leu Ser Val Glu Ala Ile Ser Ala Arg Glu Tyr Leu Lys

100 105 110

Phe Lys Glu Met Ile Phe Asp Glu Asp Trp Ala Lys Asp Asp Asn Ala

115 120 125

Leu Glu Val Asp Phe Gly Ala Phe Asp Tyr Ser Asn Pro Arg Leu Ala

130 135 140

Leu Ser Ser Ser Val Gly Asn Gly Leu Asn Phe Ile Ser Lys Val Leu

145 150 155 160

Ser Ser Lys Phe Gly Gly Lys Pro Glu Asp Ala Gln Pro Leu Leu Asp

165 170 175

Tyr Leu Leu Ala Leu Asn His Gln Gly Glu Asn Leu Met Ile Asn Glu

180 185 190

Asn Leu Asn Gly Val Ala Lys Leu Gln Ala Ala Leu Ile Val Ala Glu

195 200 205

Val Phe Val Ser Ser Phe Pro Lys Asp Thr Pro Tyr Lys Asp Phe Glu

210 215 220

His Lys Leu Lys Glu Trp Gly Phe Asp Lys Gly Trp Gly His Asn Ala

225 230 235 240

Gly Arg Val Arg Glu Thr Met Arg Leu Leu Ser Glu Ile Ile Gln Ala

245 250 255

Pro Asp Pro Ile Asn Met Glu Ser Phe Phe Ser Lys Leu Pro Thr Thr

260 265 270

Phe Asn Ile Val Ile Phe Ser Ile His Gly Tyr Phe Gly Gln Ala Asp

275 280 285

Val Leu Gly Leu Pro Asp Thr Gly Gly Gln Val Val Tyr Ile Leu Asp

290 295 300

Gln Val Arg Ala Leu Glu Glu Glu Met Leu Gln Arg Ile Lys Gln Gln

305 310 315 320

Gly Leu Asn Val Lys Pro Lys Ile Leu Val Val Ser Arg Leu Ile Pro

325 330 335

Asp Ala Arg Gly Thr Thr Cys Asn Gln Glu Met Glu Pro Ile Leu Asn

340 345 350

Ser Ser His Ser His Ile Leu Arg Ile Pro Phe Arg Thr Glu Lys Gly

355 360 365

Val Leu Arg Gln Trp Asp Ala Ser Ala Lys Ile Leu Glu Leu Met Glu

370 375 380

Gly Lys Pro Asp Leu Ile Ile Gly Asn Tyr Thr Asp Gly Asn Leu Val

385 390 395 400

Ala Ser Leu Leu Ala Asn Lys Leu Gly Val Thr Gln Gly Thr Ile Ala

405 410 415

His Ala Leu Glu Lys Thr Lys Tyr Glu Asp Ser Asp Val Lys Trp Lys

420 425 430

Gln Phe Asp Pro Lys Tyr His Phe Ser Cys Gln Phe Thr Ala Asp Leu

435 440 445

Leu Ala Met Asn Ala Ala Asp Phe Ile Ile Thr Ser Thr Tyr Gln Glu

450 455 460

Ile Ala Gly Ser Glu Thr Arg Pro Gly Gln Tyr Glu Ser His Thr Ala

465 470 475 480

Phe Thr Met Pro Gly Leu Tyr Arg Ala Val Ser Gly Ile Asn Val Phe

485 490 495

Asp Pro Lys Phe Asn Ile Ala Ala Pro Gly Ala Glu Gln Ser Thr Tyr

500 505 510

Phe Pro Phe Thr Glu Lys Gln Lys Arg Phe Ser Thr Phe Arg Pro Ala

515 520 525

Ile Asn Glu Leu Leu Tyr Ser Asn Glu Glu Asn Asn Glu His Ile Gly

530 535 540

Phe Leu Ala Asp Arg Lys Lys Pro Ile Ile Phe Ser Met Ala Arg Phe

545 550 555 560

Asp Thr Val Lys Asn Leu Ser Gly Leu Thr Glu Trp Tyr Gly Lys Asn

565 570 575

Lys Lys Leu Arg Asn Leu Val Asn Leu Val Ile Val Gly Gly Phe Phe

580 585 590

Asp Pro Ser Lys Ser Lys Asp Arg Glu Glu Ala Ala Glu Ile Lys Lys

595 600 605

Met His Glu Leu Ile Glu Lys Tyr Gln Leu Lys Gly Gln Met Arg Trp

610 615 620

Ile Ala Ala Gln Thr Asp Lys Tyr Arg Asn Ser Glu Leu Tyr Arg Thr

625 630 635 640

Ile Ala Asp Thr Lys Gly Ala Phe Val Gln Pro Ala Leu Tyr Glu Ala

645 650 655

Phe Gly Leu Thr Val Ile Glu Ala Met Asp Cys Gly Leu Pro Thr Phe

660 665 670

Ala Thr Asn Gln Gly Gly Pro Ala Glu Ile Ile Val Asp Gly Val Ser

675 680 685

Gly Phe His Ile Asp Pro Tyr Asn Gly Asp Glu Ser Ser Lys Lys Ile

690 695 700

Ala Asp Phe Phe Glu Lys Cys Lys Val Asp Ser Lys Tyr Trp Asn Arg

705 710 715 720

Ile Ser Glu Gly Gly Leu Lys Arg Ile Glu Glu Cys Tyr Thr Trp Lys

725 730 735

Ile Tyr Ala Asn Lys Val Leu Asn Met Gly Ser Ile Tyr Gly Phe Trp

740 745 750

Arg Gln Phe Asn Val Gly Gln Lys Gln Ala Lys Gln Arg Tyr Phe Glu

755 760 765

Met Phe Tyr Asn Pro Leu Phe Arg Lys Leu Ala Lys Ser Val Pro Ile

770 775 780

Pro His Glu Glu Pro Leu Pro Leu Ala Thr Ser Asp Ser Thr Gln Ser

785 790 795 800

Gln Glu Leu Lys Leu Pro Leu Pro Val Pro Ala Ala Val Ala Lys Val

805 810 815

Leu Pro Leu Thr Arg His Ala Phe Asn Leu Ile Thr Ser Leu Pro Arg

820 825 830

Val Thr Gly Lys Val Asp Val Lys

835 840

<210> 19

<211> 3268

<212> DNA

<213> Nicotiana tabacum

<400> 19

atggcctcaa ctgttgctgg tagcatgcct gatgctttga aacaaagccg atatcatatg 60

aagagatgct tcgctaggtg aacacccttc ttgttctttt tgttttttcc ctctaccatt 120

tatgtcaaat ttcaatgcat aatgctaact actttttttc tttttgactt caaaattgga 180

cgtgaaaggt tcattgcaat gggaaggagg ttgatgaagc tgaaacattt aacagaagaa 240

atagaaaaaa ctattgaaga caaggcagaa agaaccaaga ttttggaggg ttcacttgga 300

aaaattatga gttccacaca ggtcagcacc atttaaccaa cttaattgaa taggaagaaa 360

420

gctgttgtcc caccttatgt tgcttttgca gtaaggcaca atcctggctt ctgggattat 480

gtcaaagttg acgctgaaac tctctctgtg gaagctattt cagccaggga ctatctcaaa 540

ttcaaagaga tgatctttga tgaagattgg taactggaag attgtatcat tttaaagaaa 600

caatttttta atattcaaga ttagttttga tggttgaatg tgcaagcagg gcaaaggatg 660

aaaatgcact cgaagtagat tttggtgctt ttgactactc taatcatcgg ttagcccttt 720

cctcttctgt cggaaatggg ctaaacttca tctcgaaagt tttgtcttca aagtttggtg 780

gaaaggcaga agatgcccag cctttgcttg attacttact agctcttaat catcaaggag 840

aggtatggaa atggactacc ttccttttctt aaggaattat ataatgatgt atgttataaa 900

gatccttttt aaacattgac actttgcaga atctaatgat caatgagaat ctgaatggcg 960

tctctaagct tcaagcagca ttgatagtag ctgaagtttt tgtatcttcc tttcccaaag 1020

acacacctta taaagacttt gagcataagt aagcttttca aacgcttctg ttatcatatg 1080

1140

aaatactagg ctcaaagaat ggggctttga gaaagggtgg ggtcacaatg caggaagagt 1200

1260

gtcctttttc agcaggcttc ctactacatt caacattgtt atcttctcca ttcatggtta 1320

ctttggccaa gcagatgtcc ttggtttgcc cgatactgga ggccaggttt acatacacag 1380

caatttatct ccttttgcct catatttact tattagcgac acttgcatta ttgaaatcac 1440

1500

1560

gcaaaaatat gcgtagccaa ggttttgaaa ttgttcagag gggattaaga tgatcgagat 1620

atttgtttcc ttcttccatt gatgtgtaca ggtcactcgt ctcattccag atgctcgagg 1680

gactacatgc aatcaggaga tggaacctat acttaactcg tcccattctc acatcctgag 1740

aattccattc aggacagaga aggagttct tcgccaatgg gtttctcggt ttgatatcta 1800

tccttacttg gagaactatg ccaaggcaag tctcctacca aaattaccac ctattcatac 1860

actttattca gttttttgag ctaatcattc tcatttgtca cgtatgtgat taggatgctt 1920

ctgctaagat acttgagctc atggaaggta aaccagacct cattattggg aactacactg 1980

atggaaattt agtggcatct ctattggcca acaaacttgg agttactcag gttctacagc 2040

tgatcattta tctgatcaga ttttctacat tgttttcttg ataattaaac ggaaatctta 2100

tgagattgta acattttagg gaaccattgc tcatgcatta gagaaaacca agtatgaaga 2160

ttctgatgtc aagtggaagc agtttgattc caagtaccac ttttcttgcc aattcactgc 2220

cgatttattg gcaatgaatg ctgctgattt tatcattacc agcacatatc aagaaatcgc 2280

aggaaggtta gcactgactc tctcagtata tttggcaact taatgaatgt actgcttgtg 2340

gccaacacta aaagctatta ctcgtccttc agcgaaacta ggcctggaca atatgaaagt 2400

2460

ccaaagttca acattgctgc tcctggggct gaacagtctg cctatttccc cttcactgag 2520

aaacagaaac gattcagcgc gtttcgtcct gctattgagg aactacttta cagtaatgag 2580

caaaacaacg agcacatgta agtctaattg ccccattttc ctaatctaac cattgcttaa 2640

atgttctgtt tttacttgat atgtggtact tatcagtgat attttttatt ggaacagtgg 2700

2760

gaacttgtca ggcttgactg agtggtatgg gaagaataag aagttgcgga acttggttaa 2820

cctcgttatc gttgggggat tcttcgatcc atcaaaatca aaagaccgggg aggaagcagc 2880

tgaaatcaag aagatgcatg aattgattga gaaatacaag ctcaagggac aaatgagatg 2940

gatagcagct caaactgata aatatcaaaa cagtgagcta tatcgaacta ttgctgacac 3000

3060

aatgaattgt ggactgccta catttgctac taatcaaggc ggacctgcag aaatcattgt 3120

tgatggggtt tcaggcttcc atattgatcc ttacaatggg gatgaatcga gcaagaaaat 3180

3240

aggtctcaag cgcattgaag aatggtaa 3268

<210> 20

<211> 732

<212> PROTEIN

<213> Nicotiana tabacum

<400> 20

Met Ala Ser Thr Val Ala Gly Ser Met Pro Asp Ala Leu Lys Gln Ser

1 5 10 15

Arg Tyr His Met Lys Arg Cys Phe Ala Arg Phe Ile Ala Met Gly Arg

20 25 30

Arg Leu Met Lys Leu Lys His Leu Thr Glu Glu Ile Glu Lys Thr Ile

35 40 45

Glu Asp Lys Ala Glu Arg Thr Lys Ile Leu Glu Gly Ser Leu Gly Lys

50 55 60

Ile Met Ser Ser Thr Gln Glu Ala Ala Val Val Pro Pro Tyr Val Ala

65 70 75 80

Phe Ala Val Arg His Asn Pro Gly Phe Trp Asp Tyr Val Lys Val Asp

85 90 95

Ala Glu Thr Leu Ser Val Glu Ala Ile Ser Ala Arg Asp Tyr Leu Lys

100 105 110

Phe Lys Glu Met Ile Phe Asp Glu Asp Trp Ala Lys Asp Glu Asn Ala

115 120 125

Leu Glu Val Asp Phe Gly Ala Phe Asp Tyr Ser Asn His Arg Leu Ala

130 135 140

Leu Ser Ser Ser Val Gly Asn Gly Leu Asn Phe Ile Ser Lys Val Leu

145 150 155 160

Ser Ser Lys Phe Gly Gly Lys Ala Glu Asp Ala Gln Pro Leu Leu Asp

165 170 175

Tyr Leu Leu Ala Leu Asn His Gln Gly Glu Asn Leu Met Ile Asn Glu

180 185 190

Asn Leu Asn Gly Val Ser Lys Leu Gln Ala Ala Leu Ile Val Ala Glu

195 200 205

Val Phe Val Ser Ser Phe Pro Lys Asp Thr Pro Tyr Lys Asp Phe Glu

210 215 220

His Lys Leu Lys Glu Trp Gly Phe Glu Lys Gly Trp Gly His Asn Ala

225 230 235 240

Gly Arg Val Arg Glu Thr Met Arg Leu Leu Ser Glu Ile Ile Gln Ala

245 250 255

Pro Asp Pro Ile Asn Met Glu Ser Phe Phe Ser Arg Leu Pro Thr Thr

260 265 270

Phe Asn Ile Val Ile Phe Ser Ile His Gly Tyr Phe Gly Gln Ala Asp

275 280 285

Val Leu Gly Leu Pro Asp Thr Gly Gly Gln Val Val Tyr Ile Leu Asp

290 295 300

Gln Val Arg Ala Leu Glu Glu Glu Met Leu Gln Arg Ile Lys Gln Gln

305 310 315 320

Gly Leu Asn Val Lys Pro Lys Ile Leu Val Val Thr Arg Leu Ile Pro

325 330 335

Asp Ala Arg Gly Thr Thr Cys Asn Gln Glu Met Glu Pro Ile Leu Asn

340 345 350

Ser Ser His Ser His Ile Leu Arg Ile Pro Phe Arg Thr Glu Lys Gly

355 360 365

Val Leu Arg Gln Trp Asp Ala Ser Ala Lys Ile Leu Glu Leu Met Glu

370 375 380

Gly Lys Pro Asp Leu Ile Ile Gly Asn Tyr Thr Asp Gly Asn Leu Val

385 390 395 400

Ala Ser Leu Leu Ala Asn Lys Leu Gly Val Thr Gln Gly Thr Ile Ala

405 410 415

His Ala Leu Glu Lys Thr Lys Tyr Glu Asp Ser Asp Val Lys Trp Lys

420 425 430

Gln Phe Asp Ser Lys Tyr His Phe Ser Cys Gln Phe Thr Ala Asp Leu

435 440 445

Leu Ala Met Asn Ala Ala Asp Phe Ile Ile Thr Ser Thr Tyr Gln Glu

450 455 460

Ile Ala Gly Ser Glu Thr Arg Pro Gly Gln Tyr Glu Ser His Thr Ala

465 470 475 480

Phe Thr Met Pro Gly Leu Tyr Arg Ala Val Ser Gly Ile Asn Val Phe

485 490 495

Asp Pro Lys Phe Asn Ile Ala Ala Pro Gly Ala Glu Gln Ser Ala Tyr

500 505 510

Phe Pro Phe Thr Glu Lys Gln Lys Arg Phe Ser Ala Phe Arg Pro Ala

515 520 525

Ile Glu Glu Leu Leu Tyr Ser Asn Glu Gln Asn Asn Glu His Ile Gly

530 535 540

Phe Leu Ala Asp Arg Lys Lys Pro Ile Ile Phe Ser Met Ala Arg Phe

545 550 555 560

Asp Thr Val Lys Asn Leu Ser Gly Leu Thr Glu Trp Tyr Gly Lys Asn

565 570 575

Lys Lys Leu Arg Asn Leu Val Asn Leu Val Ile Val Gly Gly Phe Phe

580 585 590

Asp Pro Ser Lys Ser Lys Asp Arg Glu Glu Ala Ala Glu Ile Lys Lys

595 600 605

Met His Glu Leu Ile Glu Lys Tyr Lys Leu Lys Gly Gln Met Arg Trp

610 615 620

Ile Ala Ala Gln Thr Asp Lys Tyr Gln Asn Ser Glu Leu Tyr Arg Thr

625 630 635 640

Ile Ala Asp Thr Lys Gly Ala Phe Val Gln Pro Ala Leu Tyr Glu Ala

645 650 655

Phe Gly Leu Thr Val Ile Glu Ala Met Asn Cys Gly Leu Pro Thr Phe

660 665 670

Ala Thr Asn Gln Gly Gly Pro Ala Glu Ile Ile Val Asp Gly Val Ser

675 680 685

Gly Phe His Ile Asp Pro Tyr Asn Gly Asp Glu Ser Ser Lys Lys Ile

690 695 700

Ala Asp Phe Phe Glu Lys Cys Lys Val Asp Ser Lys Tyr Trp Asn Lys

705 710 715 720

Ile Cys Gly Gly Gly Leu Lys Arg Ile Glu Glu Trp

725 730

<210> 21

<211> 3937

<212> DNA

<213> Nicotiana tabacum

<400> 21

atggctactg caccagccct aaatagatca gagtccatag ctgatagcat gccagaggcc 60

ttaaggcaaa gccggtacca catgaagaaa tgttttgcca agtacataga gcaaggaaag 120

aggatgatga aacttcataa cttgatggat gagttggaga aagtaattga tgatcctgct 180

gaaaggaacc atgttttgga aggcttactt ggctacatat tatgcactac aatggtatag 240

ctagattcat atgtacttat gatgccctta tattgtttcc tgatgtatta ctcttaaaac 300

cttctttgat caaatttaca ggaggctgca gttgttcctc cctacattgc ctttgccacg 360

agacagaatc ctggattctg ggaatatgtg aaagtgaatg ctaatgatct ttctgttgag 420

ggtattacag ctacagaata cttgaaattc aaggaaatga tagttgatga atgctggtat 480

agtatacgtt gcagcttatc ataccttttg tggttttata acttcaatca gaaaactcat 540

cagagttacc tttgtgtgaa catgaaatgc agggcaaaag atgaatatgc actggaaatt 600

gattttggag cagtagactt ctcaacgcct cgactgaccc tatcctcttc aattggcaat 660

ggtctcagtt atgtttccaa gtttctaact tcaaagctaa atgctacctc cgcgagtgca 720

cagtgtctgg ttgactactt gctcactttg aatcatcaag gagatgtacg tcaacaaaaa 780

tcaaactcca taagtaaact tgtcaactct aagaagaaaa aataggaaaa gaagattcac 840

gtaacaaatt ttctttatgt tcaactgcag aaactgatga tcaatgagac actcagcact 900

gtctcaaagc ttcaggctgc actggttgta gcagaagcat ctatttcctc tttaccaaca 960

gatacaccat atgagagctt tgagctaagg tgatttgttt ttttcctctac ttccctccac 1020

ttgtgccatg ctacgtagta ctaagtaact tcaattcttg taaagattca aacagtgggg 1080

ttttgagaaa ggatggggtg atacagctga aagggtcagc gacaccatga gaacactgtc 1140

tgaggtgctt caggcaccag atccattgaa cattcagaag ttctttggaa gggttccaac 1200

tgttttcaat attgtattgt tctctgtcca tggatacttt ggccaagcag atgttcttgg 1260

cttgccagac actggtggtc aggtaagcat ttaatagctt ttacatttaa cttctatgca 1320

1380

atgttttgga tcaagttgta gcttttgaag aagaaatgct acaaagaatt aaacagcagg 1440

ggctcaatat taagcctcaa attcttgtgg tgagttccta gacaatcgac gtgactatgc 1500

aattatgtag aggctgttta gaaaagttaa tatcatatgt tgattgcaca gttaacccga 1560

ctgattccgg atgcaaaagg aacaaagtgc aaccaggaac tagaaccaat caagaataca 1620

aaacattcac acatcctcag agttccattt aggacagaaa aaggagtgct taatcaatgg 1680

gtttcacgat ttgatatcta tccatatctg gagagatata ctcaggtatg tatttttata 1740

tcaaccttgc tcatcaaaga tgtgttgttt cctcaattcc atttttcccc ttggcaaaag 1800

gatgctgctg acaaaatcgt cgagctaatg gaaggcaaac ctgatctaat cattggtaac 1860

tacactgatg ggaatctagt ggcttcacta atggctagaa aacttgggat aactctggta 1920

acttttctta atcatatttg atgttgcttc ttctccaagt tagttcttaa tctccactga 1980

cctagaccat ctttgcaaca gggaactatt gctcatgctt tggagaagac aaaatatgaa 2040

gactctgaca taaaattgaa ggaactcgat ccgaagtacc acttctcttg ccaattcaca 2100

gctgatttga ttgcaatgaa ttcagcagat ttcattatca ctagcacata ccaagaaata 2160

gctggaaggt aagaattaga gctaataagt aatgcattca tatgtatttc agcatcgctc 2220

tttcaccatc atcgaataca caccactact cagtaaatgt atttgctcaa aagtttgcaa 2280

cttaatggat ctcattcttg aatgcttcaa catatgcagc aaagataaac caggacagta 2340

tgagagccat agtgcattta cccttccagg gctttacaga gttgcttcag gtatcaatgt 2400

ctttgatcca aaatttaata ttgctgcacc tggggcagac cagtcggtgt atttccctta 2460

cacagaaaag cagaagcgtt tgactgcttt ccgccctgcc attgaggaac tgctttttag 2520

taaagtggac aatgacgagc acgtgtaagt ctaagtgtta aacttcagct tagtgcctag 2580

aacatcccac tgctctatgt attgatgttt cacttgtttc aaacagtgga tatttagaag 2640

acagaaagaa acctatcctg tttaccatgg caaggctgga cacagtgaag aacacatctg 2700

gactaacaga atggtatggc aagaacaaga ggctcagaag cttagttaac cttgttgtgg 2760

ttggtggttc ctttgatcct acaaaatcca aggataggga agaagcagct gaaataaaaa 2820

agatgcacat gctgatagag aaataccagc ttaagggtca gattagatgg atagcagctc 2880

agactgacag atacagaaat agtgaactct accgcacaat agcagattcc aaaggagctt 2940

ttgtgcagcc tgcattgtat gaagcatttg gtctaacagt cattgaggca atgaactgtg 3000

gattaccaac ctttgctacc aaccaaggtg gccctgctga gattattgtt gatggggtct 3060

caggctttca tattgatcca aataatgggg atgaatcaag caacaaaatt gccaactttt 3120

tccaaaaatg cagggaggat cctgagtatt ggaacaggat ttcagtccag ggtctaaacc 3180

gtatatatga atggtaactc acagataagc cattcaaatt gcaaagaggc acatatcttg 3240

cagaaaattt cttaatcctt aaatcctaat ttttgcagt tacacatgga agatctatgc 3300

3360

gaaacaagca aagcaaagat acatcgagac tttctacaat cttgagttta ggaacttggt 3420

atagtgctgc atgacattga cagtatacca caaacatctt tatgagatga attactttta 3480

ataaaattgt ttttaacctt tgcttcctta atggcactta ttgcaggtaa aaaatgtgcc 3540

gacgaaacac cacaaggacc aaaggagagg gagaaagtta agccacagat 3600

3660

tagtactaaa ttagaattat acagcatgca gcttttgctg ttcacctttc taaatcacca 3720

gttgtgtcaa tcaagttgac aaaatcaata aattgggatt ttccctttcc tatgcttgat 3780

3840

ttactacaac tgtactgaca tactaattat ttctgtgtac caggcgctca caatcaaggt 3900

tgcagaagta agattagata aaattgctac tgcatga 3937

<210> 22

<211> 860

<212> PROTEIN

<213> Nicotiana tabacum

<400> 22

Met Ala Thr Ala Pro Ala Leu Asn Arg Ser Glu Ser Ile Ala Asp Ser

1 5 10 15

Met Pro Glu Ala Leu Arg Gln Ser Arg Tyr His Met Lys Lys Cys Phe

20 25 30

Ala Lys Tyr Ile Glu Gln Gly Lys Arg Met Met Lys Leu His Asn Leu

35 40 45

Met Asp Glu Leu Glu Lys Val Ile Asp Asp Pro Ala Glu Arg Asn His

50 55 60

Val Leu Glu Gly Leu Leu Gly Tyr Ile Leu Cys Thr Thr Met Glu Ala

65 70 75 80

Ala Val Val Pro Pro Tyr Ile Ala Phe Ala Thr Arg Gln Asn Pro Gly

85 90 95

Phe Trp Glu Tyr Val Lys Val Asn Ala Asn Asp Leu Ser Val Glu Gly

100 105 110

Ile Thr Ala Thr Glu Tyr Leu Lys Phe Lys Glu Met Ile Val Asp Glu

115 120 125

Cys Trp Ala Lys Asp Glu Tyr Ala Leu Glu Ile Asp Phe Gly Ala Val

130 135 140

Asp Phe Ser Thr Pro Arg Leu Thr Leu Ser Ser Ser Ile Gly Asn Gly

145 150 155 160

Leu Ser Tyr Val Ser Lys Phe Leu Thr Ser Lys Leu Asn Ala Thr Ser

165 170 175

Ala Ser Ala Gln Cys Leu Val Asp Tyr Leu Leu Thr Leu Asn His Gln

180 185 190

Gly Asp Lys Leu Met Ile Asn Glu Thr Leu Ser Thr Val Ser Lys Leu

195 200 205

Gln Ala Ala Leu Val Val Ala Glu Ala Ser Ile Ser Ser Leu Pro Thr

210 215 220

Asp Thr Pro Tyr Glu Ser Phe Glu Leu Arg Phe Lys Gln Trp Gly Phe

225 230 235 240

Glu Lys Gly Trp Gly Asp Thr Ala Glu Arg Val Ser Asp Thr Met Arg

245 250 255

Thr Leu Ser Glu Val Leu Gln Ala Pro Asp Pro Leu Asn Ile Gln Lys

260 265 270

Phe Phe Gly Arg Val Pro Thr Val Phe Asn Ile Val Leu Phe Ser Val

275 280 285

His Gly Tyr Phe Gly Gln Ala Asp Val Leu Gly Leu Pro Asp Thr Gly

290 295 300

Gly Gln Val Val Tyr Val Leu Asp Gln Val Val Ala Phe Glu Glu Glu

305 310 315 320

Met Leu Gln Arg Ile Lys Gln Gln Gly Leu Asn Ile Lys Pro Gln Ile

325 330 335

Leu Val Leu Thr Arg Leu Ile Pro Asp Ala Lys Gly Thr Lys Cys Asn

340 345 350

Gln Glu Leu Glu Pro Ile Lys Asn Thr Lys His Ser His Ile Leu Arg

355 360 365

Val Pro Phe Arg Thr Glu Lys Gly Val Leu Asn Gln Trp Val Ser Arg

370 375 380

Phe Asp Ile Tyr Pro Tyr Leu Glu Arg Tyr Thr Gln Asp Ala Ala Asp

385 390 395 400

Lys Ile Val Glu Leu Met Glu Gly Lys Pro Asp Leu Ile Ile Gly Asn

405 410 415

Tyr Thr Asp Gly Asn Leu Val Ala Ser Leu Met Ala Arg Lys Leu Gly

420 425 430

Ile Thr Leu Gly Thr Ile Ala His Ala Leu Glu Lys Thr Lys Tyr Glu

435 440 445

Asp Ser Asp Ile Lys Leu Lys Glu Leu Asp Pro Lys Tyr His Phe Ser

450 455 460

Cys Gln Phe Thr Ala Asp Leu Ile Ala Met Asn Ser Ala Asp Phe Ile

465 470 475 480

Ile Thr Ser Thr Tyr Gln Glu Ile Ala Gly Ser Lys Asp Lys Pro Gly

485 490 495

Gln Tyr Glu Ser His Ser Ala Phe Thr Leu Pro Gly Leu Tyr Arg Val

500 505 510

Ala Ser Gly Ile Asn Val Phe Asp Pro Lys Phe Asn Ile Ala Ala Pro

515 520 525

Gly Ala Asp Gln Ser Val Tyr Phe Pro Tyr Thr Glu Lys Gln Lys Arg

530 535 540

Leu Thr Ala Phe Arg Pro Ala Ile Glu Glu Leu Leu Phe Ser Lys Val

545 550 555 560

Asp Asn Asp Glu His Val Gly Tyr Leu Glu Asp Arg Lys Lys Pro Ile

565 570 575

Leu Phe Thr Met Ala Arg Leu Asp Thr Val Lys Asn Thr Ser Gly Leu

580 585 590

Thr Glu Trp Tyr Gly Lys Asn Lys Arg Leu Arg Ser Leu Val Asn Leu

595 600 605

Val Val Val Gly Gly Ser Phe Asp Pro Thr Lys Ser Lys Asp Arg Glu

610 615 620

Glu Ala Ala Glu Ile Lys Lys Met His Met Leu Ile Glu Lys Tyr Gln

625 630 635 640

Leu Lys Gly Gln Ile Arg Trp Ile Ala Ala Gln Thr Asp Arg Tyr Arg

645 650 655

Asn Ser Glu Leu Tyr Arg Thr Ile Ala Asp Ser Lys Gly Ala Phe Val

660 665 670

Gln Pro Ala Leu Tyr Glu Ala Phe Gly Leu Thr Val Ile Glu Ala Met

675 680 685

Asn Cys Gly Leu Pro Thr Phe Ala Thr Asn Gln Gly Gly Pro Ala Glu

690 695 700

Ile Ile Val Asp Gly Val Ser Gly Phe His Ile Asp Pro Asn Asn Gly

705 710 715 720

Asp Glu Ser Ser Asn Lys Ile Ala Asn Phe Phe Gln Lys Cys Arg Glu

725 730 735

Asp Pro Glu Tyr Trp Asn Arg Ile Ser Val Gln Gly Leu Asn Arg Ile

740 745 750

Tyr Glu Cys Tyr Thr Trp Lys Ile Tyr Ala Asn Lys Val Leu Asn Met

755 760 765

Gly Ser Ile Tyr Thr Phe Trp Arg Thr Leu Tyr Arg Asp Gln Lys Gln

770 775 780

Ala Lys Gln Arg Tyr Ile Glu Thr Phe Tyr Asn Leu Glu Phe Arg Asn

785 790 795 800

Leu Val Lys Asn Val Pro Ile Arg Lys Asp Glu Thr Pro Gln Gly Pro

805 810 815

Lys Glu Arg Glu Lys Val Lys Pro Gln Ile Ser Gln Arg His Ala Leu

820 825 830

Lys Leu Leu Pro Thr Val Phe Gln Glu Thr Leu Ala Leu Thr Ile Lys

835 840 845

Val Ala Glu Val Arg Leu Asp Lys Ile Ala Thr Ala

850 855 860

<210> 23

<211> 3905

<212> DNA

<213> Nicotiana tabacum

<400> 23

atggctactg caccagccct gaaaagatca gagtccatag ctgatagcat gccagaggcc 60

ttaaggcaaa gccggtacca catgaagaaa tgttttgcca agtacataga gcaaggcaag 120

aggatgatga aacttcataa cttgatggat gaattggaga aagtaattga tgatcctgct 180

gaaaggaacc atgttttgga aggcttactt ggctacatat tatgtactac aatggtatag 240

ctagattcat atgtacttat gatgtcctta tattgtttcc ggaggcatta ttcttaaatc 300

cttctttgat caaatttgta ggaggctgca gttgttcctc cctatattgc cttcgccacg 360

agacagaatc ctggattctg ggaatatgtg aaagtcaatg ctaatgatct ttctgttgag 420

ggtattacag ctacagatta cttgaaattc aaggaaatga tagttgatga aagctggtat 480

agaatacttt gcagcttatc ataccttttg tggttttata atttcaatca gaaaactcat 540

cagagttacc tttgtgtgaa catgacatgc agggcaaaag atgaatatgc actggaaatt 600

gattttggag cagtagactt ctcaacgcct cgactgaccc tatcctcttc aattggaaat 660

ggtctcagtt atgtttccaa gtttctaact tcaaagctaa atgctacctc agcgagtgca 720

cagtgtctgg ttgactactt gctcactttg aatcaccaag gagatgtacg tcaacaaaaa 780

tcaaactcca taagtaaact tgtcaactct aagaagtaaa aataggaaaa gaagattcat 840

gtaacaaatt ttctttatgt tcaactgtag aaactgatga tcaatgagac actcggcact 900

gtctcaaagc ttcaggctgc actggttgta gcagaagcat ctatttcctc cttaccaaca 960

gatacaccat accagagctt tgagctaagg tgatttgttt ttttcctctac ttccttccac 1020

ttttggtgtg ctacatagta ctaagtaact tcaattcttg taaagattca aacagtgggg 1080

ttttgagaaa ggatggggtg atacagctga aagggtccgc gacaccatga gaacactttc 1140

tgaggtactt caggcgccag atccattgaa cattgagaag ttctttggga gggttccaac 1200

tgttttcaat attgtattgt tctctgttca tggatacttt ggccaagcaa atgttcttgg 1260

cttgccagac acaggtggtc aggtaagcat ctaatagctt ttacatttaa cttctatgca 1320

1380

cttgttcaac aggtggttta tgttttggat caagttgtag cttttgaaga agaaatgctc 1440

caaagaatta aacagcaggg gctcaatatt aagcctcaaa ttcttgtggt gagctcctag 1500

acaatgacgt gactatgcaa ttaagtagag gctgtttaga aaagttaata tcatatgttg 1560

attgcacagt taacccgact gattccggac gccaaaggaa caaagtgcaa cggaacta 1620

gaaccaatca agaatacaaa acattcacac atcctcagag ttccatttag gacagaaaaa 1680

ggagtgctta atcaatgggt ttcacgattt gatatctatc catatctgga gagatatact 1740

caggtgtgta tttttatc aaccctgctc atcaaagatg tgttgtttcc tcaattccat 1800

ttttcgcctt gacaaaagga cgctgctgac aaaatcatcg agctaatgga aggcaaacct 1860

gatctaatca ttggtaacta cactgatggg aatctagtgg cttctctaat ggctagaaag 1920

cttgggataa ctctggtaac ttttcttatc atatttgatg ttgtttcttc tccaagttgg 1980

ttcttaatgt caactaaccc agaccatctt tgtaacaggg aactattgct catgctctgg 2040

agaagacaaa atatgaagac tctgacatca aattgaagga actcgatccg aagtaccact 2100

tttcttgcca attcacagct gatttgattg caatgaattc agcagatttc attatcacaa 2160

2220

agtagtgcac tcatatattt cagcatcgct ctttcgcata atcgaataca caccactact 2280

cagtaaatgt acttgctcaa aagtttacaa gtttatggat cttattcttg aatgcttcaa 2340

catatgcagc aaagataggc caggacagta tgagagccat agtgcattta cccttccagg 2400

gctttacaga gttgcttcag gcatcaatgt ctttgatcct aaatttaata ttgctgcacc 2460

tggggcagac caatcggtgt atttccctta cacagaaaag cagacgcgtt tgactgcttt 2520

ccgccctgcc attgaggaac tgctttttag taaagtggac aatgacgagc acatgtaagt 2580

cttagtgtta aacttcagct ttcagcttag tgcctagaac attccactgg ctctatgtat 2640

taatgtttca cttgtttcaa acacagtgga tatttagaag acagaaagaa acctatcctg 2700

tttaccatgg caaggctgga cacagtgaag aacacatctg gactaacaga atggtatggc 2760

aagaacaaga ggctcagaag cttagttaac cttgttgtgg ttggtggttc ctttgatcct 2820

acaaaatcca aggatagaga agaagcagct gaaataaaaa agatgcacat gctgatagag 2880

aaataccagc ttaagggtca gatcagatgg atagcagctc agactgacag atatagaaac 2940

agtgaactct accgcacaat agcagattcc aaaggagctt ttgtgcagcc tgcattatat 3000

gaagcatttg gtctaacagt cattgaggca atgaactgtg gattaccaac ctttgctacc 3060

aaccaaggtg gccctgctga gattattgtt gatggggtct caggctttca tattgatcca 3120

aataatgggg atgaatcaag caacaaagtt gccaactttt tccaaaaatg cagggaggat 3180

cctgagtatt ggaacaggat ttcagtccag ggtctaaacc gtatatatga atggtaactc 3240

acagataagc cattcaaatt gcaaagaggc acatatcttg ctgaaaattt cttaatcctt 3300

3360

ggtccatcta tactttttgg aggacattgt acagagatca gaaacaagca aagcaaagat 3420

acatcgagac tttctacaat cttgagttta ggaacttggt atagtgctgc atgacattga 3480

3540

tgcctcctta atgacactta ttgcaggtaa aaaatgtgcc tatcagacag gacgaaacac 3600

3660

taaagctttt gcctatagtt tttcaggaga ccctagtata ttctagtact aaattagaat 3720

tatacagcat gcagcttgct tctgctgttc acctttctaa atcaccagtt atgtcaatca 3780

3840

acttcgttta tggtagtctt ccttcattgt tttctcctgt acttctttta ctacaactgt 3900

actga 3905

<210> 24

<211> 913

<212> PROTEIN

<213> Nicotiana tabacum

<400> 24

Met Ala Thr Ala Pro Ala Leu Lys Arg Ser Glu Ser Ile Ala Asp Ser

1 5 10 15

Met Pro Glu Ala Leu Arg Gln Ser Arg Tyr His Met Lys Lys Cys Phe

20 25 30

Ala Lys Tyr Ile Glu Gln Gly Lys Arg Met Met Lys Leu His Asn Leu

35 40 45

Met Asp Glu Leu Glu Lys Val Ile Asp Asp Pro Ala Glu Arg Asn His

50 55 60

Val Leu Glu Gly Leu Leu Gly Tyr Ile Leu Cys Thr Thr Met Glu Ala

65 70 75 80

Ala Val Val Pro Pro Tyr Ile Ala Phe Ala Thr Arg Gln Asn Pro Gly

85 90 95

Phe Trp Glu Tyr Val Lys Val Asn Ala Asn Asp Leu Ser Val Glu Gly

100 105 110

Ile Thr Ala Thr Asp Tyr Leu Lys Phe Lys Glu Met Ile Val Asp Glu

115 120 125

Ser Trp Ala Lys Asp Glu Tyr Ala Leu Glu Ile Asp Phe Gly Ala Val

130 135 140

Asp Phe Ser Thr Pro Arg Leu Thr Leu Ser Ser Ser Ile Gly Asn Gly

145 150 155 160

Leu Ser Tyr Val Ser Lys Phe Leu Thr Ser Lys Leu Asn Ala Thr Ser

165 170 175

Ala Ser Ala Gln Cys Leu Val Asp Tyr Leu Leu Thr Leu Asn His Gln

180 185 190

Gly Asp Lys Leu Met Ile Asn Glu Thr Leu Gly Thr Val Ser Lys Leu

195 200 205

Gln Ala Ala Leu Val Val Ala Glu Ala Ser Ile Ser Ser Leu Pro Thr

210 215 220

Asp Thr Pro Tyr Gln Ser Phe Glu Leu Arg Phe Lys Gln Trp Gly Phe

225 230 235 240

Glu Lys Gly Trp Gly Asp Thr Ala Glu Arg Val Arg Asp Thr Met Arg

245 250 255

Thr Leu Ser Glu Val Leu Gln Ala Pro Asp Pro Leu Asn Ile Glu Lys

260 265 270

Phe Phe Gly Arg Val Pro Thr Val Phe Asn Ile Val Leu Phe Ser Val

275 280 285

His Gly Tyr Phe Gly Gln Ala Asn Val Leu Gly Leu Pro Asp Thr Gly

290 295 300

Gly Gln Val Val Tyr Val Leu Asp Gln Val Val Ala Phe Glu Glu Glu

305 310 315 320

Met Leu Gln Arg Ile Lys Gln Gln Gly Leu Asn Ile Lys Pro Gln Ile

325 330 335

Leu Val Leu Thr Arg Leu Ile Pro Asp Ala Lys Gly Thr Lys Cys Asn

340 345 350

Gln Glu Leu Glu Pro Ile Lys Asn Thr Lys His Ser His Ile Leu Arg

355 360 365

Val Pro Phe Arg Thr Glu Lys Gly Val Leu Asn Gln Trp Val Ser Arg

370 375 380

Phe Asp Ile Tyr Pro Tyr Leu Glu Arg Tyr Thr Gln Asp Ala Ala Asp

385 390 395 400

Lys Ile Ile Glu Leu Met Glu Gly Lys Pro Asp Leu Ile Ile Gly Asn

405 410 415

Tyr Thr Asp Gly Asn Leu Val Ala Ser Leu Met Ala Arg Lys Leu Gly

420 425 430

Ile Thr Leu Gly Thr Ile Ala His Ala Leu Glu Lys Thr Lys Tyr Glu

435 440 445

Asp Ser Asp Ile Lys Leu Lys Glu Leu Asp Pro Lys Tyr His Phe Ser

450 455 460

Cys Gln Phe Thr Ala Asp Leu Ile Ala Met Asn Ser Ala Asp Phe Ile

465 470 475 480

Ile Thr Ser Thr Tyr Gln Glu Ile Ala Gly Ser Lys Asp Arg Pro Gly

485 490 495

Gln Tyr Glu Ser His Ser Ala Phe Thr Leu Pro Gly Leu Tyr Arg Val

500 505 510

Ala Ser Gly Ile Asn Val Phe Asp Pro Lys Phe Asn Ile Ala Ala Pro

515 520 525

Gly Ala Asp Gln Ser Val Tyr Phe Pro Tyr Thr Glu Lys Gln Thr Arg

530 535 540

Leu Thr Ala Phe Arg Pro Ala Ile Glu Glu Leu Leu Phe Ser Lys Val

545 550 555 560

Asp Asn Asp Glu His Ile Gly Tyr Leu Glu Asp Arg Lys Lys Pro Ile

565 570 575

Leu Phe Thr Met Ala Arg Leu Asp Thr Val Lys Asn Thr Ser Gly Leu

580 585 590

Thr Glu Trp Tyr Gly Lys Asn Lys Arg Leu Arg Ser Leu Val Asn Leu

595 600 605

Val Val Val Gly Gly Ser Phe Asp Pro Thr Lys Ser Lys Asp Arg Glu

610 615 620

Glu Ala Ala Glu Ile Lys Lys Met His Met Leu Ile Glu Lys Tyr Gln

625 630 635 640

Leu Lys Gly Gln Ile Arg Trp Ile Ala Ala Gln Thr Asp Arg Tyr Arg

645 650 655

Asn Ser Glu Leu Tyr Arg Thr Ile Ala Asp Ser Lys Gly Ala Phe Val

660 665 670

Gln Pro Ala Leu Tyr Glu Ala Phe Gly Leu Thr Val Ile Glu Ala Met

675 680 685

Asn Cys Gly Leu Pro Thr Phe Ala Thr Asn Gln Gly Gly Pro Ala Glu

690 695 700

Ile Ile Val Asp Gly Val Ser Gly Phe His Ile Asp Pro Asn Asn Gly

705 710 715 720

Asp Glu Ser Ser Asn Lys Val Ala Asn Phe Phe Gln Lys Cys Arg Glu

725 730 735

Asp Pro Glu Tyr Trp Asn Arg Ile Ser Val Gln Gly Leu Asn Arg Ile

740 745 750

Tyr Glu Cys Tyr Thr Trp Lys Ile Tyr Ala Asn Lys Val Leu Asn Met

755 760 765

Gly Ser Ile Tyr Thr Phe Trp Arg Thr Leu Tyr Arg Asp Gln Lys Gln

770 775 780

Ala Lys Gln Arg Tyr Ile Glu Thr Phe Tyr Asn Leu Glu Phe Arg Asn

785 790 795 800

Leu Val Lys Asn Val Pro Ile Arg Gln Asp Glu Thr Pro Gln Gly Pro

805 810 815

Lys Glu Arg Arg Glu Lys Val Lys Pro Gln Ile Ser Gln Arg His Ala

820 825 830

Leu Lys Leu Leu Pro Ile Val Phe Gln Glu Thr Leu Val Tyr Ser Ser

835 840 845

Thr Lys Leu Glu Leu Tyr Ser Met Gln Leu Ala Ser Ala Val His Leu

850 855 860

Ser Lys Ser Pro Val Met Ser Ile Lys Leu Thr Lys Ser Ile Asn Ser

865 870 875 880

Ala Phe Pro Phe Pro Met Leu Asp Cys Tyr Tyr Ser Tyr Phe Val Tyr

885 890 895

Gly Ser Leu Pro Ser Leu Phe Ser Pro Val Leu Leu Leu Leu Gln Leu

900 905 910

Tyr

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Claims (28)

1. Dried plant material from Nicotiana tabacum for use in the preparation of a tobacco product, comprising: (i) a polynucleotide consisting of a sequence having at least 95% sequence identity with SEQ ID NO: 9 or SEQ ID NO: 11; or a polynucleotide consisting of a sequence having at least 80% sequence identity with SEQ ID NO: 5 or SEQ ID NO: 13; (ii) a polypeptide encoded by the polynucleotide specified in (i); (iii) a polypeptide consisting of a sequence having at least 95% sequence identity with SEQ ID NO: 6 or SEQ ID NO: 14; or at least 93% sequence identity with SEQ ID NO: 10 or SEQ ID NO: 12; or (iv) a construct, vector or expression vector containing the isolated polynucleotide referred to in (i), wherein said plant material contains at least one modification that modulates the expression or activity of the polynucleotide or polypeptide as compared to that of a control dried plant material in which the expression or activity of the polynucleotide or polypeptide is unmodified, and wherein the modulated expression or activity of the polynucleotide or polypeptide modulates the level of reducing sugars in the dried plant material compared to the level of reducing sugars in the control dried plant material, and wherein at least one modification is a modification of the genome of the dried plant material or a modification of a construct, vector or expression vector or a transgenic modification, and/or wherein at least one modification is an interference polynucleotide containing a sequence that is complementary to at least 19 nucleotides of the RNA transcribed from the polynucleotide (i). 2. The plant material according to claim 1, wherein the modification of the genome of the dried plant material or the modification of the construct, vector or expression vector is a mutation or editing. 3. Plant material according to any one of the preceding claims, wherein the modification increases or decreases the expression or activity of the polynucleotide or polypeptide compared to the control dried plant material. 4. Plant material according to any one of the preceding claims, wherein the reducing sugar is glucose or fructose. 5. Plant material according to any one of the preceding claims, containing biomass, seed, stem, flowers or leaves. 6. Plant material according to any one of the preceding claims, which has been fire-dried, sun-dried or air-dried. 7. Plant material according to any one of the preceding claims subjected to homogenization. 8. Tobacco product containing dried plant material according to any one of the preceding claims. 9. The method of obtaining dried plant material from Nicotiana tabacum according to any one of paragraphs. 1-7 containing (a) providing a cell of a Nicotiana tabacum plant containing a polynucleotide consisting of a sequence having at least 95% sequence identity with SEQ ID NO: 9 or SEQ ID NO: 11; or characterized by at least 80% sequence identity with SEQ ID NO: 5 or SEQ ID NO: 13; (b) modifying the plant cell to modulate the expression of the polynucleotide as compared to that in the control plant cell; (c) regenerating the plant cell to obtain a plant; (d) collection of plant material from it; And (f) drying the plant material. 10. The method of claim 9, wherein the step of modifying the plant cell includes modifying the genome of the cell using genome editing or genome engineering techniques; preferably, wherein genome editing or genomic engineering techniques are selected from CRISPR/Cas technology, zinc finger nuclease mediated mutagenesis, chemical or radiation mutagenesis, homologous recombination, oligonucleotide directed mutagenesis, and meganuclease mediated mutagenesis. 11. The method according to claim 9 or 10, in which the step of modifying a plant cell includes transfecting the cell with a construct containing a polynucleotide consisting of a sequence characterized by at least 95% sequence identity with SEQ ID NO: 9, or SEQ ID NO: 11; or characterized by at least 80% sequence identity with SEQ ID NO: 5 or SEQ ID NO: 13, functionally linked to a constitutive promoter; and/or wherein the plant cell is transfected with a construct expressing an interference polynucleotide containing a sequence that is complementary to at least 19 nucleotides of RNA transcribed from a polynucleotide consisting of a sequence characterized by at least 95% sequence identity with SEQ ID NO: 9 or SEQ ID NO: 11; or a polynucleotide consisting of a sequence having at least 80% sequence identity with SEQ ID NO: 5 or SEQ ID NO: 13. 12. The method according to any one of paragraphs. 9-11, wherein the plant material comprises dried leaves, dried stems, or dried flowers, or a mixture thereof; and/or wherein the drying method is selected from the group consisting of air drying, fire drying, flue drying, and fire tube drying.